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Sample records for adenoviral oncoprotein e1b55k

  1. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    International Nuclear Information System (INIS)

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-01-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells

  2. E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression.

    Science.gov (United States)

    Müncheberg, Sarah; Hay, Ron T; Ip, Wing H; Meyer, Tina; Weiß, Christina; Brenke, Jara; Masser, Sawinee; Hadian, Kamyar; Dobner, Thomas; Schreiner, Sabrina

    2018-04-25

    HAdV E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in non-permissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 Ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established.RNF4, a cellular SUMO-targeted Ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM, and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNAi resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor, which was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 Ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain productive viral life

  3. E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus▿

    Science.gov (United States)

    Thomas, Michael A.; Broughton, Robin S.; Goodrum, Felicia D.; Ornelles, David A.

    2009-01-01

    Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function. PMID:19129452

  4. E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus.

    Science.gov (United States)

    Thomas, Michael A; Broughton, Robin S; Goodrum, Felicia D; Ornelles, David A

    2009-03-01

    Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

  5. KAP1 Is a Host Restriction Factor That Promotes Human Adenovirus E1B-55K SUMO Modification

    DEFF Research Database (Denmark)

    Bürck, Carolin; Mund, Andreas; Berscheminski, Julia

    2016-01-01

    Once transported to the replication sites, HAdVs need to assure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly characte......Once transported to the replication sites, HAdVs need to assure decondensation and transcriptional activation of their viral genomes to synthesize viral proteins and initiate steps to reprogram the host cell for viral replication. These early stages during adenoviral infection are poorly...... characterized, but represent a decisive moment in establishing a productive infection. Here, we identify a novel host viral restriction factor, KAP1. This heterochromatin associated transcription factor regulates the dynamic organization of host chromatin structure via its ability to influence epigenetic marks...

  6. The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

    Directory of Open Access Journals (Sweden)

    Jasdave S Chahal

    Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

  7. Presumed Acute Adenoviral Dacryoadenitis Associated with ...

    African Journals Online (AJOL)

    of serum adenoviral antibodies IgM and presence of inclusion bodies in conjunctival smear. Epidemic keratoconjunctivitis is caused by adenovirus serovars. 8, 19, and 37.[7] Serological typing and polymerase chain reaction for adenoviral were not available in smear in both eyes. A computerized tomography scan of.

  8. Genetically engineering adenoviral vectors for gene therapy.

    Science.gov (United States)

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  9. Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

    Science.gov (United States)

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel J; Kalle, Wouter H J

    2005-06-01

    Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

  10. Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

    National Research Council Canada - National Science Library

    Huang, Shuang

    2004-01-01

    .... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

  11. Oncoprotein metastasis and its suppression revisited

    Directory of Open Access Journals (Sweden)

    Radulescu Razvan T

    2010-04-01

    Full Text Available Abstract The past two decades have witnessed an increasing appreciation of the role of the tumor microenvironment, of genetic and epigenetic alterations in normal cells adjacent to tumors and of the migration of normal cells with aberrant intrinsic properties in cancer pathophysiology. Aside from these insights, a novel concept termed "oncoprotein metastasis" (OPM has recently been advanced and proposed to reflect protein-based neoplastic phenomena that might occur even before any modifications relating to the morphology, location or (epigenetic outfit of cells during the malignant process. Here, evidence is presented that supports the OPM perception and thus should contribute not only to further rethink the definition of a normal cell, but also the treatment of cancer disease in the years to come.

  12. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    NARCIS (Netherlands)

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

    2003-01-01

    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer

  13. Reactivation of presumed adenoviral keratitis after laser in situ keratomileusis.

    Science.gov (United States)

    Safak, Nilgün; Bilgihan, Kamil; Gürelik, Gökhan; Ozdek, Sengül; Hasanreisoğlu, Berati

    2002-04-01

    We report a patient with reactivation of presumed adenoviral keratoconjunctivitis after laser in situ keratomileusis (LASIK) to correct high myopia. The preoperative refraction was -13.00 diopters (D) in the right eye and -14.00 D in the left eye, and the best corrected visual acuity was 20/20 in both eyes. On the first postoperative day, mild conjunctival hyperemia and multiple subepithelial infiltrations localized in the flap zone consistent with adenoviral keratoconjunctivitis were seen. After prompt treatment, the lesions resolved. As a consequence, LASIK successfully corrected the high myopia. Adenoviral keratoconjunctivitis can be reactivated after LASIK, unlike after photorefractive keratectomy, despite the absence of symptomatic and clinical findings before the procedure.

  14. Adenoviral vectors as genome editing tools : repairing defective DMD alleles

    NARCIS (Netherlands)

    Maggio, Ignazio

    2016-01-01

    Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as carriers of designer nucleases and donor DNA sequences, the research presented in this thesis expands the utility of

  15. Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

    OpenAIRE

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determine...

  16. Molecular epidemiology of adenoviral keratoconjunctivitis in Saudi Arabia.

    Science.gov (United States)

    Tabbara, Khalid F; Omar, Nazri; Hammouda, Ehab; Akanuma, Masataka; Ohguchi, Takeshi; Ariga, Toshihide; Tagawa, Yoshitsugu; Kitaichi, Nobuyoshi; Ishida, Susumu; Aoki, Koki; Ishiko, Hiroaki; Ohno, Shigeaki

    2010-10-24

    Adenoviral keratoconjunctivitis is a major cause of ocular morbidity and may lead to visual loss. Adenovirus types 8, 19, and 37 may cause epidemic keratoconjunctivitis. The main objective of this study was to determine the types of adenoviruses causing keratoconjunctivitis in Saudi Arabia. We conducted a non-interventional observational clinical study. Seventy three eyes from 65 patients who presented to The Eye Center in Riyadh, Saudi Arabia with clinical features of acute adenoviral keratoconjunctivitis were included. Each patient underwent complete clinical examination and features such as membranous reaction, conjunctival hemorrhage, subepithelial corneal infiltrates, and preauricular lymph node enlargement were recorded. Conjunctival swabs were obtained from patients with presumed acute viral conjunctivitis. Immunochromatography (IC) and restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) were performed on the conjunctival swabs obtained from each eye. Serotype identification was performed using direct sequencing technique. Forty-nine (67.1%) were adenovirus type 8, 8 (11.0%) were adenovirus type 3, 6 (8.2%) type 37, 5 (6.8%) were adenovirus type 4, and 2 (2.3%) type 19. The remaining 5 were types 14, 19, and 22. The prevalence of membranous conjunctivitis was highest (83%) among eyes with adenovirus type 37 while subepithelial corneal opacities were most commonly seen among eyes with adenovirus type 8 (47%). Immunochromatography tests were positive for adenovirus in 48 (65.7%) out of 73 eyes. This study determined the types of adenoviruses causing keratoconjunctivitis at one center in Saudi Arabia. Direct sequencing techniques is an efficient, accurate, and rapid means of diagnosing adenoviral keratoconjunctivitis. The most common causes of adenoviral keratoconjunctivitis in Saudi Arabia were adenovirus types 8, 3, and 37. Membranous conjunctivitis and subepithelial opacities had the highest frequency of adenovirus types 37 and 8

  17. ETS-1 oncoprotein expression is decreased in aggressive papillary ...

    African Journals Online (AJOL)

    So far, there is no reliable prognostic marker has been proved for detection of the tumor progression and recurrence. Objectives: To analyze the correlation between ETS-1 oncoprotein immunohistochemical expression and the different stages and grades of the primary papillary transitional cell carcinoma of the urinary ...

  18. Gene therapy for barrett's esophagus: adenoviral gene transfer in different intestinal models

    NARCIS (Netherlands)

    Marsman, Willem A.; Buskens, Christianne J.; Wesseling, John G.; van Lanschot, J. Jan B.; Bosma, Piter J.

    2005-01-01

    Adenoviral gene therapy could potentially be used for treatment of patients with a Barrett's esophagus. In order to study the feasibility of this approach it is important to study adenoviral intestinal transduction both in vitro and in vivo. In the present study, we used differentiating Caco-2

  19. ERG oncoprotein expression in prostate carcinoma patients of different ethnicities

    OpenAIRE

    KELLY, GREGORY M.; KONG, YINK HEAY; DOBI, ALBERT; SRIVASTAVA, SHIV; SESTERHENN, ISABELL A.; PATHMANATHAN, RAJADURAI; TAN, HUI MENG; TAN, SHYH-HAN; CHEONG, SOK CHING

    2014-01-01

    Overexpression of the erythroblast transformation-specific-related gene (ERG) oncoprotein due to transmembrane protease, serine 2 (TMPRSS2)-ERG fusion, the most prevalent genomic alteration in prostate cancer (CaP), is more frequently observed among Caucasian patients compared to patients of African or Asian descent. To the best of our knowledge, this is the first study to investigate the prevalence of ERG alterations in a multiethnic cohort of CaP patients. A total of 191 formalin-fixed para...

  20. An Adenoviral Vector Based Vaccine for Rhodococcus equi.

    Directory of Open Access Journals (Sweden)

    Carla Giles

    Full Text Available Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.

  1. Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

    Science.gov (United States)

    Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

  2. Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

    Science.gov (United States)

    Lopez-Gordo, Estrella; Podgorski, Iva I; Downes, Nicholas; Alemany, Ramon

    2014-04-01

    Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.

  3. Adenoviral vector immunity: its implications and circumvention strategies.

    Science.gov (United States)

    Ahi, Yadvinder S; Bangari, Dinesh S; Mittal, Suresh K

    2011-08-01

    Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations.

  4. Adenoviral hemorrhagic disease in California mule deer, 1990-2014.

    Science.gov (United States)

    Woods, Leslie W; Schumaker, Brant A; Pesavento, Patricia A; Crossley, Beate M; Swift, Pamela K

    2018-03-01

    We reviewed case records from the California Animal Health and Food Safety (CAHFS) laboratory and the California Department of Fish and Wildlife (CDFW) spanning 25 years (1990-2014) for all deer accessions submitted to CAHFS for pathology and/or histopathology, with and without a diagnosis of adenoviral hemorrhagic disease (AHD), in order to determine the prevalence of AHD in California. We also examined spatial and temporal distribution, age, and mule deer subspecies in deer that died from AHD. Of 483 deer submitted to CAHFS for diagnostic testing in 1990-2014, 17.2% were diagnosed with confirmed AHD, and 26.5% were confirmed plus suspected cases of AHD. Columbian black-tailed deer ( Odocoileus hemionus columbianus), particularly fawns and juveniles, were most frequently affected. Deer adenovirus ( Odocoileus adenovirus 1; OdAdV-1) was detected by immunohistochemistry in archived CDFW formalin-fixed, paraffin-embedded tissues from deer that died in mortality events in 1981, 1983, and 1986-1987. OdAdV-1 is a common cause of hemorrhagic disease mortality events in California deer, and mortality as a result of AHD is documented as early as 1981.

  5. Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

    Energy Technology Data Exchange (ETDEWEB)

    Ugai, Hideyo; Dobbins, George C.; Wang, Minghui [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Le, Long P. [Massachusetts General Hospital, Pathology Service, 55 Fruit St.-GRJ 249, Boston, MA 02114 (United States); Matthews, David A. [School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, Bristol BS8 1TD (United Kingdom); Curiel, David T., E-mail: dcuriel@radonc.wustl.edu [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2012-10-25

    Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

  6. Suppression of inflammation by dexamethasone prolongs adenoviral vector-mediated transgene expression in the facial nucleus of the rat

    NARCIS (Netherlands)

    Hermens, W.T.J.M.C.; Verhaagen, J

    1998-01-01

    Adenoviral vector directed gene transfer to rat facial motoneurons occurs efficiently following intra-parenchymal injection of relatively high dosages (> or =10(7) pfu per injection) of a prototype first generation adenoviral vector. However, high level of transgene expression, as observed during

  7. Transient gene transfer to neurons and glia : analysis of adenoviral vector performance in the CNS and PNS

    NARCIS (Netherlands)

    Hermens, W.T.J.M.C.; Giger, Roman J; Holtmaat, Anthony J D G; Dijkhuizen, Paul A; Houweling, D A; Verhaagen, J

    In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied,

  8. Efficient adenoviral vector directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

    NARCIS (Netherlands)

    Gispen, W.H.; Holtmaat, A.J.G.D.; Hermens, W.T.J.M.C.; Oestreicher, A.B.; Kaplitt, M.G.; Verhaagen, J.

    1996-01-01

    Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that

  9. Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

    NARCIS (Netherlands)

    Holtmaat, Anthony J D G; Hermens, W.T.J.M.C.; Oestreicher, A B; Gispen, Willem Hendrik; Kaplitt, M G; Verhaagen, J

    1996-01-01

    Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that

  10. Adenoviral vector-mediated gene transfer and neurotransplantation : possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus

    NARCIS (Netherlands)

    van Esseveldt, K E; Liu, R.; Hermens, W.T.J.M.C.; Verhaagen, J; Boer, G J

    Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural

  11. Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

    Science.gov (United States)

    Buttgereit, P; Weineck, S; Röpke, G; Märten, A; Brand, K; Heinicke, T; Caselmann, W H; Huhn, D; Schmidt-Wolf, I G

    2000-08-01

    Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and

  12. Viral oncoprotein antibodies as a marker for recurrence of Merkel cell carcinoma: A prospective validation study.

    Science.gov (United States)

    Paulson, Kelly G; Lewis, Christopher W; Redman, Mary W; Simonson, William T; Lisberg, Aaron; Ritter, Deborah; Morishima, Chihiro; Hutchinson, Kathleen; Mudgistratova, Lola; Blom, Astrid; Iyer, Jayasri; Moshiri, Ata S; Tarabadkar, Erica S; Carter, Joseph J; Bhatia, Shailender; Kawasumi, Masaoki; Galloway, Denise A; Wener, Mark H; Nghiem, Paul

    2017-04-15

    Merkel cell carcinoma (MCC) is an aggressive skin cancer with a recurrence rate of >40%. Of the 2000 MCC cases per year in the United States, most are caused by the Merkel cell polyomavirus (MCPyV). Antibodies to MCPyV oncoprotein (T-antigens) have been correlated with MCC tumor burden. The present study assesses the clinical utility of MCPyV-oncoprotein antibody titers for MCC prognostication and surveillance. MCPyV-oncoprotein antibody detection was optimized in a clinical laboratory. A cohort of 219 patients with newly diagnosed MCC were followed prospectively (median follow-up, 1.9 years). Among the seropositive patients, antibody titer and disease status were serially tracked. Antibodies to MCPyV oncoproteins were rare among healthy individuals (1%) but were present in most patients with MCC (114 of 219 patients [52%]; P < .01). Seropositivity at diagnosis independently predicted decreased recurrence risk (hazard ratio, 0.58; P = .04) in multivariate analyses adjusted for age, sex, stage, and immunosuppression. After initial treatment, seropositive patients whose disease did not recur had rapidly falling titers that became negative by a median of 8.4 months. Among seropositive patients who underwent serial evaluation (71 patients; 282 time points), an increasing oncoprotein titer had a positive predictive value of 66% for clinically evident recurrence, whereas a decreasing titer had a negative predictive value of 97%. Determination of oncoprotein antibody titer assists in the clinical management of patients with newly diagnosed MCC by stratifying them into a higher risk seronegative cohort, in which radiologic imaging may play a more prominent role, and into a lower risk seropositive cohort, in which disease status can be tracked in part by oncoprotein antibody titer. Cancer 2017;123:1464-1474. © 2016 American Cancer Society. © 2016 American Cancer Society.

  13. Adenoviral DNA replication: DNA sequences and enzymes required for initiation in vitro

    International Nuclear Information System (INIS)

    Stillman, B.W.; Tamanoi, F.

    1983-01-01

    In this paper evidence is provided that the 140,000-dalton DNA polymerase is encoded by the adenoviral genome and is required for the initiation of DNA replication in vitro. The DNA sequences in the template DNA that are required for the initiation of replication have also been identified, using both plasmid DNAs and synthetic oligodeoxyribonucleotides. 48 references, 7 figures, 1 table

  14. Ovarian cancer targeted adenoviral-mediated mda-7/IL-24 gene therapy

    NARCIS (Netherlands)

    Mahasreshti, PJ; Kataram, M; Wu, HJ; Yalavarthy, LP; Carey, D; Dent, P; Chada, S; Alvarez, RD; Haisma, HJ; Fisher, PB; Curiel, DT

    Objective. We have previously shown that adenoviral-mediated melanoma differentiation-associated gene-7 (Ad.mda-7) therapy induces apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7. The objective of this study was to

  15. Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells

    NARCIS (Netherlands)

    Gijsbers, Linda; Koel, Björn; Weggeman, Miranda; Goudsmit, Jaap; Havenga, Menzo; Marzio, Giuseppe

    2005-01-01

    Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for

  16. ERG oncoprotein expression in prostate carcinoma patients of different ethnicities.

    Science.gov (United States)

    Kelly, Gregory M; Kong, Yink Heay; Dobi, Albert; Srivastava, Shiv; Sesterhenn, Isabell A; Pathmanathan, Rajadurai; Tan, Hui Meng; Tan, Shyh-Han; Cheong, Sok Ching

    2015-01-01

    Overexpression of the erythroblast transformation-specific-related gene (ERG) oncoprotein due to transmembrane protease, serine 2 ( TMPRSS2 ) -ERG fusion, the most prevalent genomic alteration in prostate cancer (CaP), is more frequently observed among Caucasian patients compared to patients of African or Asian descent. To the best of our knowledge, this is the first study to investigate the prevalence of ERG alterations in a multiethnic cohort of CaP patients. A total of 191 formalin-fixed paraffin-embedded sections of transrectal ultrasound-guided prostate biopsy specimens, collected from 120 patients treated at the Sime Darby Medical Centre, Subang Jaya, Malaysia, were analyzed for ERG protein expression by immunohistochemistry using the anti-ERG monoclonal antibody 9FY as a surrogate for the detection of ERG fusion events. The overall frequency of ERG protein expression in the population evaluated in this study was 39.2%. Although seemingly similar to rates reported in other Asian communities, the expression of ERG was distinct amongst different ethnic groups (P=0.004). Malaysian Indian (MI) patients exhibited exceedingly high expression of ERG in their tumors, almost doubling that of Malaysian Chinese (MC) patients, whereas ERG expression was very low amongst Malay patients (12.5%). When collectively analyzing data, we observed a significant correlation between younger patients and higher ERG expression (P=0.04). The prevalence of ERG expression was significantly different amongst CaP patients of different ethnicities. The higher number of ERG-expressing tumors among MI patients suggested that the TMPRSS2-ERG fusion may be particularly important in the pathogenesis of CaP amongst this group of patients. Furthermore, the more frequent expression of ERG among the younger patients analyzed suggested an involvement of ERG in the early onset of CaP. The results of this study underline the value of using ERG status to better understand the differences in the

  17. Positive selection on a bacterial oncoprotein associated with gastric cancer

    Directory of Open Access Journals (Sweden)

    Delgado-Rosado Gisela

    2011-11-01

    Full Text Available Background Helicobacter pylori is a vertically inherited gut commensal that is carcinogenic if it possesses the cag pathogenicity island (cag PaI; infection with H.pylori is the major risk factor for gastric cancer, the second leading cause of death from cancer worldwide (WHO. The cag PaI locus encodes the cagA gene, whose protein product is injected into stomach epithelial cells via a Type IV secretion system, also encoded by the cag PaI. Once there, the cagA protein binds to various cellular proteins, resulting in dysregulation of cell division and carcinogenesis. For this reason, cagA may be described as an oncoprotein. A clear understanding of the mechanism of action of cagA and its benefit to the bacteria is lacking. Results Here, we reveal that the cagA gene displays strong signatures of positive selection in bacteria isolated from amerindian populations, using the Ka/Ks ratio. Weaker signatures are also detected in the gene from bacteria isolated from asian populations, using the Ka/Ks ratio and the more sensitive branches-sites model of the PAML package. When the cagA gene isolated from amerindian populations was examined in more detail it was found that the region under positive selection contains the EPIYA domains, which are known to modulate the carcinogenicity of the gene. This means that the carcinogenicity modulating region of the gene is undergoing adaptation. The results are discussed in relation to the high incidences of stomach cancer in some latin american and asian populations. Conclusion Positive selection on cagA indicates antagonistic coevolution between host and bacteria, which appears paradoxical given that cagA is detrimental to the human host upon which the bacteria depends. This suggests several non-exclusive possibilities; that gastric cancer has not been a major selective pressure on human populations, that cagA has an undetermined benefit to the human host, or that horizontal transmission of H.pylori between hosts

  18. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    International Nuclear Information System (INIS)

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-01-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  19. Targeting of the MUC1-C Oncoprotein in Colitis-Associated Colorectal Cancer

    Science.gov (United States)

    2014-11-01

    separated by infiltrates of inflammatory cells and the presence of crypt abscess (lower left panel). With progression to dysplasia, the crypts are...Rajabi, H, et al., MUC1-C oncoprotein induces TCF7L2 activation and promotes cyclin D1 expression in human breast cancer cells. J Biol Chem, 2012

  20. Peptide-DNA conjugates as tailored bivalent binders of the oncoprotein c-Jun.

    Science.gov (United States)

    Pazos, Elena; Portela, Cecilia; Penas, Cristina; Vázquez, M Eugenio; Mascareñas, José L

    2015-05-21

    We describe a ds-oligonucleotide-peptide conjugate that is able to efficiently dismount preformed DNA complexes of the bZIP regions of oncoproteins c-Fos and c-Jun (AP-1), and therefore might be useful as disrupters of AP-1-mediated gene expression pathways.

  1. The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells.

    Science.gov (United States)

    Shi, Hui; Fang, Runping; Li, Yinghui; Li, Leilei; Zhang, Weiying; Wang, Huawei; Chen, Fuquan; Zhang, Shuqin; Zhang, Xiaodong; Ye, Lihong

    2016-11-28

    Hepatitis B X-interacting protein (HBXIP) as an oncoprotein plays crucial roles in the development of cancer, involving glucose metabolism reprogramming. In this study, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis in liver cancer. Here, we showed that the expression level of phosphoenolpyruvate carboxykinase (PCK1), a key enzyme of gluconeogenesis, was lower in clinical hepatocellular carcinoma (HCC) tissues than that in normal tissues. Mechanistically, HBXIP inhibited the expression of PCK1 through down-regulating transcription factor FOXO1 in hepatoma cells, and up-regulated miR-135a targeting the 3'UTR of FOXO1 mRNA in the cells. In addition, HBXIP increased the phosphorylation levels of FOXO1 protein by activating PI3K/Akt pathway, leading to the export of FOXO1 from nucleus to cytoplasm. Strikingly, over-expression of PCK1 could abolish the HBXIP-promoted growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Our finding provides new insights into the mechanism by which oncoprotein HBXIP modulates glucose metabolism reprogramming in HCC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Problem-Solving Test: The Mechanism of Action of a Human Papilloma Virus Oncoprotein

    Science.gov (United States)

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: human papilloma virus; cervical cancer; oncoproteins; malignant transformation; retinoblastoma protein; cell cycle; quiescent and cycling cells; cyclin/cyclin-dependent kinase (Cdk) complexes; E2F; S-phase genes; enhancer element; proto-oncogenes; tumor suppressor genes; radioactive…

  3. Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer

    Science.gov (United States)

    Sen, Prakriti; Ganguly, Pooja; Ganguly, Niladri

    2018-01-01

    Human papillomaviruses (HPVs) are double stranded circular DNA viruses that infect cutaneous and mucosal epithelial cells. Almost 99% of cervical cancer has a HPV infection. The early oncoproteins E6 and E7 are important in this cellular transformation process. Epigenetic mechanisms have long been known to result in decisive alterations in DNA, leading to alterations in DNA-protein interactions, alterations in chromatin structure and compaction and significant alterations in gene expression. The enzymes responsible for these epigenetic modifications are DNA methyl transferases (DNMTs), histone acetylases and deacetylases. Epigenetics has an important role in cancer development by modifying the cellular micro environment. In this review, the authors discuss the role of HPV oncoproteins E6 and E7 in modulating the epigenetic mechanisms inside the host cell. The oncoproteins induce the expression of DNMTs which lead to aberrant DNA methylations and disruption of the normal epigenetic processes. The E7 oncoprotein may additionally directly bind and induce methyl transferase activity of the enzyme. These modulations lead to altered gene expression levels, particularly the genes involved in apoptosis, cell cycle and cell adhesion. In addition, the present review discusses how epigenetic mechanisms may be targeted for possible therapeutic interventions for HPV mediated cervical cancer. PMID:29285184

  4. Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Philip Ng

    2010-09-01

    Full Text Available Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application.

  5. Isolation of proteins involved in the replication of adenoviral DNA in vitro

    International Nuclear Information System (INIS)

    Lichy, J.H.; Nagata, K.; Friefeld, B.R.; Enomoto, T.; Field, J.; Guggenheimer, R.A.; Ikeda, J.E.; Horwitz, M.S.; Hurwitz, J.

    1983-01-01

    The simple mechanism of replication of adenoviral DNA has made adenovirus an especially useful model system for studies of eukaryotic replication mechanisms. The availability of this in vitro system that replicates exogenously added Ad DNA-pro has made it possible to characterize the factors involved in replication. The results presented in this paper summarize our further fractionation of the in vitro system. First, the properties of two factors purified from the uninfected nuclear extract are described. Second, the separation of the pTP/Ad Pol complex into subunits and the properties of the isolated subunits are presented. The 140K protein is shown to possess the Ad DNA polymerase activity. The results suggest that the only DNA polymerase required for adenoviral DNA replication in vitro is the 140K Ad DNA polymerase and that this enzyme is probably a viral gene product. 50 references, 10 figures, 3 tables

  6. Treatment for Retinopathy of Prematurity in an Infant with Adenoviral Conjunctivitis

    Directory of Open Access Journals (Sweden)

    Murat Gunay

    2015-01-01

    Full Text Available Retinopathy of prematurity (ROP has been a major problematic disorder during childhood. Laser photocoagulation (LPC has been proven to be effective in most of the ROP cases. Adenoviral conjunctivitis (AVC is responsible for epidemics among adult and pediatric population. It has also been reported to be a cause of outbreaks in neonatal intensive care units (NICU several times. We herein demonstrate a case with AVC who underwent LPC for ROP. And we discuss the treatment methodology in such cases.

  7. When should adenoviral non-gonococcal urethritis be suspected? Two case reports.

    Science.gov (United States)

    Avolio, Manuela; De Rosa, Rita; Modolo, Maria Luisa; Stano, Paola; Camporese, Alessandro

    2014-01-01

    The impact of Adenovirus as agent of non-gonococcal urethritis (NGU) is still poorly documented in the literature. We describe two cases showing that adenoviral infection should be reasonably hypothesized in men with dysuria and scant urethral discharge in addition to meatus inflammation and/or edema (meatitis) or conjunctivitis. Case 1: a 55-year-old man came to our observation in July 2012 referring a 5-day-history of intense dysuria and scant mucoid urethral discharge. Physical examination revealed the urethral discharge referred, but also modest meatitis and an intense conjunctival hyperemia on his right eye. Adenoviral infection was investigated and Adenovirus DNA (type 37) was detected in both the urethral and conjunctival swabs. Case 2: a 43-year-old man with intense dysuria, started 4-5 days earlier, came to our attention with his wife in August 2012. Scant urethral mucoid secretions, severe meatal inflammation of the male patient were revealed during physical examination. His wife instead complained of a 2-day history of intense burning eyes. Adenoviral infection was investigated and Adenovirus DNA (type 37) was positive both in the male urethral swab and in his wife's conjunctival swab. Adenovirus seems to cause a distinct and recognisable clinical syndrome in men presenting with urethritis. Studies on the prevalence and role of Adenovirus as a causative agent of urethritis are limited. Moreover, as rapid advanced molecular microbiology is now available, we believe that extending the search to Adenovirus in sexually active men with dysuria, scant discharge in addition to meatitis or conjunctivitis, should be a useful approach improving our understanding about adenoviral NGU, and especially avoiding or stopping unnecessary empirical antibiotic therapy.

  8. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

    OpenAIRE

    Seandel, Marco; Butler, Jason M.; Kobayashi, Hideki; Hooper, Andrea T.; White, Ian A.; Zhang, Fan; Vertes, Eva L.; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V.; Hackett, Neil R.; Rabbany, Sina; Boyer, Julie L.; Rafii, Shahin

    2008-01-01

    Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates orga...

  9. Adenoviral infection in a collection of juvenile inland bearded dragons (Pogona vitticeps).

    Science.gov (United States)

    Doneley, R J T; Buckle, K N; Hulse, L

    2014-01-01

    Juvenile inland bearded dragons (Pogona vitticeps) from a breeding collection in south-east Queensland were presented at age 6-10 weeks with neurological signs, poor growth and occasional deaths. Histopathological examination revealed that six of eight lizards had multifocal non-suppurative hepatitis associated with 5-10 μm diameter, smudgy, basophilic, hyaline intranuclear inclusion bodies that marginated the nuclear chromatin. These histological lesions were considered consistent with adenoviral hepatitis. Infection with adenovirus was confirmed positive in one of the eight dragons by PCR for adenoviral DNA. DNA was extracted from formalin-fixed paraffin-embedded pooled tissues of the juvenile inland bearded dragons and tested using a nested-PCR protocol with primers specific for identification of adenovirus. Sequencing of the one PCR-positive dragon showed 95% nucleotide sequence alignment with agamid atadenovirus 1. Further investigation involved testing the breeding population, including the parents of the affected juveniles. Blood and cloacal samples were collected from the adult population, DNA was extracted and tested by PCR for adenovirus. There was a high percentage of positive results from the samples collected from the breeding population. This is the first reported group outbreak of adenoviral disease in bearded dragons in Australia. © 2014 Australian Veterinary Association.

  10. Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

    Directory of Open Access Journals (Sweden)

    Jianfeng Zhang

    2012-11-01

    Full Text Available The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future.

  11. Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

    Science.gov (United States)

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Kalle, Wouter H J

    2004-11-01

    Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

  12. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    International Nuclear Information System (INIS)

    Manzo-Merino, Joaquin; Massimi, Paola; Lizano, Marcela; Banks, Lawrence

    2014-01-01

    The HPV-16 E6 and E6 ⁎ proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6 ⁎ . Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8

  13. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    Energy Technology Data Exchange (ETDEWEB)

    Manzo-Merino, Joaquin [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Massimi, Paola [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy); Lizano, Marcela, E-mail: lizanosoberon@gmail.com [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Banks, Lawrence, E-mail: banks@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy)

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  14. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

    OpenAIRE

    Puil, L; Liu, J; Gish, G; Mbamalu, G; Bowtell, D; Pelicci, P G; Arlinghaus, R; Pawson, T

    1994-01-01

    The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interacti...

  15. Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats : preferential survival of transduced astroglial cells in nude rats

    NARCIS (Netherlands)

    Hermens, W.T.J.M.C.; Verhaagen, J

    1997-01-01

    In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of

  16. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector.

    Science.gov (United States)

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-10-31

    Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation.

  17. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector

    Directory of Open Access Journals (Sweden)

    Ando Tomoaki

    2005-10-01

    Full Text Available Abstract Genetic deficiency in the expression of interleukin-10 (IL-10 is associated with the onset and progression of experimental inflammatory bowel disease (IBD. The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10, an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate, a common model of colitis. Adenoviral IL-10 (Ad-IL10 transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS, Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10 gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation.

  18. Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography.

    Science.gov (United States)

    Eglon, Marc N; Duffy, Aoife M; O'Brien, Timothy; Strappe, Padraig M

    2009-11-01

    Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

  19. Sensitivity and specificity of the AdenoPlus test for diagnosing adenoviral conjunctivitis.

    Science.gov (United States)

    Sambursky, Robert; Trattler, William; Tauber, Shachar; Starr, Christopher; Friedberg, Murray; Boland, Thomas; McDonald, Marguerite; DellaVecchia, Michael; Luchs, Jodi

    2013-01-01

    To compare the clinical sensitivity and specificity of the AdenoPlus test with those of both viral cell culture (CC) with confirmatory immunofluorescence assay (IFA) and polymerase chain reaction (PCR) at detecting the presence of adenovirus in tear fluid. A prospective, sequential, masked, multicenter clinical trial enrolled 128 patients presenting with a clinical diagnosis of acute viral conjunctivitis from a combination of 8 private ophthalmology practices and academic centers. Patients were tested with AdenoPlus, CC-IFA, and PCR to detect the presence of adenovirus. The sensitivity and specificity of AdenoPlus were assessed for identifying cases of adenoviral conjunctivitis. Of the 128 patients enrolled, 36 patients' results were found to be positive by either CC-IFA or PCR and 29 patients' results were found to be positive by both CC-IFA and PCR. When compared only with CC-IFA, AdenoPlus showed a sensitivity of 90% (28/31) and specificity of 96% (93/97). When compared only with PCR, AdenoPlus showed a sensitivity of 85% (29/34) and specificity of 98% (89/91). When compared with both CC-IFA and PCR, AdenoPlus showed a sensitivity of 93% (27/29) and specificity of 98% (88/90). When compared with PCR, CC-IFA showed a sensitivity of 85% (29/34) and specificity of 99% (90/91). AdenoPlus is sensitive and specific at detecting adenoviral conjunctivitis. An accurate and rapid in-office test can prevent the misdiagnosis of adenoviral conjunctivitis that leads to the spread of disease, unnecessary antibiotic use, and increased health care costs. Additionally, AdenoPlus may help a clinician make a more informed treatment decision regarding the use of novel therapeutics. clinicaltrials.gov Identifier: NCT00921895.

  20. Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

    Science.gov (United States)

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Abu-Asab, Mones S; Tsokos, Maria; Morris, John C; Kalle, Wouter H J

    2007-04-01

    We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

  1. Targeting of the MUC1-C Oncoprotein in Colitis-Associated Colorectal Cancer

    Science.gov (United States)

    2013-09-01

    effect- level isobologram analysis is shown for ED 90 (), ED 75 (+) and ED 50 (×) with the right side panel indicating the CI index for the...was generated by exposing the cells to fixed IC50 ratios of GO-203, GSK1120212 and the GO- 203/GSK1120212 combination. The multiple effect- level ...Kharbanda S, and Kufe D, MUC1 oncoprotein blocks GSK3β -mediated phosphorylation and degradation of β-catenin. Cancer Res, 2005. 65:10413-22. 11. Rajabi

  2. Radiolabeled adenoviral sub-unit proteins for molecular imaging and therapeutic applications in oncology

    International Nuclear Information System (INIS)

    Srivastava, Suresh C.

    2005-01-01

    Full text: Our group has initiated investigations on the use of radiolabeled adenoviral (Ad) sub-unit proteins for delivering suitable radionuclides into tumor cells for molecular imaging as well as for combined gene/radionuclide therapy of cancer. A number of issues involved in developing combined gene/radionuclide delivery into tumors mediated by Ad vectors have been identified and are being addressed. Whereas current clinical trials of gene therapy using Ad vectors involve non-systemic delivery of therapeutic genes, the delivery of radionuclides preferably would involve systemic (i.v.) administration. The distribution and delivery of Ad sub-unit proteins following i.v. administration is not understood and must be studied and optimized. In addition, retention of the selective binding and internalization into tumor cells of the radiolabeled viral vectors remains an unmet challenge. We used the intact adenovirus (Ad, ∼80 nm diameter), native adenoviral fiber protein (AdFP, 180 kD trimer, purified from infected human cultured cells) and the adenoviral fiber 'knob' protein (recombinant AdFKP, 60 kD, synthesized in E. Coli), all of which interact with the in-vivo cellular receptor, coxsackie and adenovirus receptor (CAR) through the knob domain of the adenovirus fiber protein. Our initial studies were aimed at optimizing the labeling conditions using I-131 and In-111 to maintain CAR binding activity of the radiolabeled preparations. The CAR-binding was retained as determined using reaction with biotinylated CAR followed by chemiluminescence detection. The biodistribution results in mice and rats following i.v. administration (autoradiography, tissue counting) showed that all three vectors localized preferentially in CAR-expressing organs (liver, lung, heart, kidney), as expected. The CAR-binding of Ad-2 wild serotype was better (∼8 x stronger) than Ad-12, in particular following radiolabeling. Based on the above results, we further focused on the recombinant knob

  3. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    International Nuclear Information System (INIS)

    Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

    2008-01-01

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

  4. Sidestream smoke exposure increases the susceptibility of airway epithelia to adenoviral infection.

    Directory of Open Access Journals (Sweden)

    Priyanka Sharma

    Full Text Available Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR.Cultured human airway epithelial cells (CaLu-3 were used as a model to investigate the effect of sidestream cigarette smoke (SSS, mainstream cigarette smoke (MSS, or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection.This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant

  5. Expression of a single, viral oncoprotein in skin epithelium is sufficient to recruit lymphocytes.

    Directory of Open Access Journals (Sweden)

    Allison Choyce

    Full Text Available Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16 E7 oncoprotein under a keratin 14 promoter (K14E7 mice display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes to premalignant skin.

  6. The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins

    Directory of Open Access Journals (Sweden)

    Ning Li

    2015-12-01

    Full Text Available Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

  7. Regulation of the Wnt/β-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins

    Directory of Open Access Journals (Sweden)

    Jesus Omar Muñoz Bello

    2015-08-01

    Full Text Available Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV-related cancers as the lesion progresses. Specifically, β-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/β-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. Viruses 2015, 7 4735 This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/β-catenin pathway.

  8. The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal

    Energy Technology Data Exchange (ETDEWEB)

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín [Programa de Doctorado en Ciencias Biomédicas, Instituto de Fisiología Celular (IFC), Universidad Nacional Autónoma de México (UNAM), Ciudad de México 04510, México (Mexico); Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Ciudad de México 07360, México (Mexico); Ocadiz-Delgado, Rodolfo; García-Villa, Enrique [Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Ciudad de México 07360, México (Mexico); Bonilla-Delgado, José [Unidad de Investigación, Hospital Juárez de México, Ciudad de México 07760, México (Mexico); Lagunas-Martínez, Alfredo [División de Biología Molecular de Patógenos, CISEI, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México (Mexico); and others

    2016-12-15

    Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E{sub 2}) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E{sub 2} on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E{sub 2} in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. -- Graphical abstract: The HPV16 E7 oncoprotein and 17β-estradiol are involved in the upregulation of Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal ability of cancer stem cells in cervical cancer. - Highlights: •The HPV16 E7 oncoprotein enhances cellular proliferation and dedifferentiation. •The E7 oncoprotein induces stemness-related genes expression in vivo and in vitro. •The 17β-estradiol induces stemness-related genes expression in vivo. •The HPV16 E7 oncoprotein is involved in the cell self-renewal of cancer cells.

  9. The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal

    International Nuclear Information System (INIS)

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Bonilla-Delgado, José; Lagunas-Martínez, Alfredo

    2016-01-01

    Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E 2 ) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E 2 on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E 2 in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. -- Graphical abstract: The HPV16 E7 oncoprotein and 17β-estradiol are involved in the upregulation of Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal ability of cancer stem cells in cervical cancer. - Highlights: •The HPV16 E7 oncoprotein enhances cellular proliferation and dedifferentiation. •The E7 oncoprotein induces stemness-related genes expression in vivo and in vitro. •The 17β-estradiol induces stemness-related genes expression in vivo. •The HPV16 E7 oncoprotein is involved in the cell self-renewal of cancer cells.

  10. Oncoprotein MDM2 Overexpression is Associated with Poor Prognosis in Distinct Non-Hodgkin's Lymphoma Entities

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    1999-01-01

    MDM2 is an oncoprotein involved in the regulation of p53. MDM2 exerts its tumorigenic potential through p53-dependent and -independent mechanisms. It is frequently overexpressed in various malignancies. Little is known about the prognostic value of MDM2 expression in non-Hodgkin's lymphomas (NHL...... overexpression was present in 42 (22%) of 188 cases. The frequency was highest in aggressive/very aggressive NHL (P lymphomas, MDM2 overexpression was associated with higher-grade disease (P = .008). MDM2 overexpression was not related to a phenotype indicating...... altered p53. In univariate analysis MDM2 overexpression associated with short survival in follicle center lymphomas (P = .0256), extranodal marginal zone lymphomas (P lymphomas (P = .0047). The relation to poor prognosis was maintained in a Cox regression analysis including known...

  11. hrHPV E5 oncoprotein: immune evasion and related immunotherapies.

    Science.gov (United States)

    de Freitas, Antonio Carlos; de Oliveira, Talita Helena Araújo; Barros, Marconi Rego; Venuti, Aldo

    2017-05-25

    The immune response is a key factor in the fight against HPV infection and related cancers, and thus, HPV is able to promote immune evasion through the expression of oncogenes. In particular, the E5 oncogene is responsible for modulation of several immune mechanisms, including antigen presentation and inflammatory pathways. Moreover, E5 was suggested as a promising therapeutic target, since there is still no effective medical therapy for the treatment of HPV-related pre-neoplasia and cancer. Indeed, several studies have shown good prospective for E5 immunotherapy, suggesting that it could be applied for the treatment of pre-cancerous lesions. Thus, insofar as the majority of cervical, oropharyngeal and anal cancers are caused by high-risk HPV (hrHPV), mainly by HPV16, the aim of this review is to discuss the immune pathways interfered by E5 oncoprotein of hrHPV highlighting the various aspects of the potential immunotherapeutic approaches.

  12. Karyopherin β3: A new cellular target for the HPV-16 E5 oncoprotein

    International Nuclear Information System (INIS)

    Krawczyk, Ewa; Hanover, John A.; Schlegel, Richard; Suprynowicz, Frank A.

    2008-01-01

    Epidemiological and experimental studies have shown that high-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer worldwide, and that HPV-16 is associated with more than half of these cases. In addition to the well-characterized E6 and E7 oncoproteins of HPV-16, recent evidence increasingly has implicated the HPV-16 E5 protein (16E5) as an important mediator of oncogenic transformation. Since 16E5 has no known intrinsic enzymatic activity, its effects on infected cells are most likely mediated by interactions with various cellular proteins and/or its documented association with lipid rafts. In the present study, we describe a new cellular target that binds to 16E5 in COS cells and in stable human ectocervical cell lines. This target is karyopherin β3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway

  13. Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

    Science.gov (United States)

    Bosse, Kristopher R; Raman, Pichai; Zhu, Zhongyu; Lane, Maria; Martinez, Daniel; Heitzeneder, Sabine; Rathi, Komal S; Kendsersky, Nathan M; Randall, Michael; Donovan, Laura; Morrissy, Sorana; Sussman, Robyn T; Zhelev, Doncho V; Feng, Yang; Wang, Yanping; Hwang, Jennifer; Lopez, Gonzalo; Harenza, Jo Lynne; Wei, Jun S; Pawel, Bruce; Bhatti, Tricia; Santi, Mariarita; Ganguly, Arupa; Khan, Javed; Marra, Marco A; Taylor, Michael D; Dimitrov, Dimiter S; Mackall, Crystal L; Maris, John M

    2017-09-11

    We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    Science.gov (United States)

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  15. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  16. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    Directory of Open Access Journals (Sweden)

    Merry ZC Ruan

    2016-01-01

    Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

  17. Efficient Gene Delivery to Pig Airway Epithelia and Submucosal Glands Using Helper-Dependent Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Huibi Cao

    2013-01-01

    Full Text Available Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF. However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR. Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.

  18. Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

    Science.gov (United States)

    Bak, Eun-Jung; Jho, Yeonsook; Woo, Gye-Hyeong

    2018-06-01

    Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

  19. Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

    International Nuclear Information System (INIS)

    Kanagawa, Naoko; Koretomo, Ryosuke; Murakami, Sayaka; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Nakagawa, Shinsaku; Fujita, Takuya; Yamamoto, Akira; Okada, Naoki

    2008-01-01

    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35ΔRGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The α v -integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-α, and interferon-α in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of α v -integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes

  20. Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells.

    Science.gov (United States)

    Byk, T; Haddada, H; Vainchenker, W; Louache, F

    1998-11-20

    Adenoviral vectors have the potential to infect a large number of cell types including quiescent cells. Their use in hematopoietic cells is limited by the episomal form of their DNA, leading to transgene loss in the progeny cells. However, the use of this vector may be interesting for short-term in vitro modifications of primitive human hematopoietic cells. Therefore, we have investigated the ability of adenovirus to transduce cord blood CD34+ cells. Several promoters were tested using the lacZ reporter gene. The PGK and CMV promoters induced transgene expression in 18-25% of the cells, whereas the HTLV-I and especially the RSV promoter were almost inactive. To improve infection efficiency, adenovirus was complexed with cationic lipids. Lipofectamine, Cellfectin, and RPR120535b, but not Lipofectin, Lipofectace, or DOTAP, markedly improved transgene expression in CD34+ cells (from 19 to 35%). Lipofectamine strongly enhanced infection efficiency of the poorly infectable primitive CD34+CD38low cells (from 11 to 28%) whereas the more mature CD34+CD38+ cells were only slightly affected (from 24 to 31%). Lipofectamine tripled the infection of CFU-GMs and LTC-ICs derived from the CD34+CD38low cell fraction (from 4 to 12% and from 5 to 16%, respectively) and doubled that of BFU-Es (from 13 to 26%). We conclude that cationic lipids can markedly increase the efficiency of adenovirus-mediated gene transfer into primitive hematopoietic cells.

  1. A molecular model for the differential activation of STAT3 and STAT6 by the herpesviral oncoprotein tip.

    Directory of Open Access Journals (Sweden)

    Eman Dey Mazumder

    Full Text Available Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2 domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.

  2. Human Papillomavirus Type 16 E6 and E7 Oncoproteins Act Synergistically to Cause Head and Neck Cancer in Mice

    Science.gov (United States)

    Jabbar, Sean; Strati, Katerina; Shin, Myeong Kyun; Pitot, Henry C.; Lambert, Paul F.

    2010-01-01

    High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer. PMID:20797753

  3. Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

    International Nuclear Information System (INIS)

    Nanda, Dharmin; Jong, Marion de; Bakker, Willem; Bijster, Magda; Cox, Peter; Vogels, Ronald; Havenga, Menzo; Driesse, Maarten; Avezaat, Cees; Morin, Kevin; Naimi, Ebrahim; Knaus, Edward; Wiebe, Leonard; Smitt, Peter Sillevis

    2002-01-01

    Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives.The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of 123 I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with 123 I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). 123 I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with 123 I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after 123 I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with the transgene in the former E1

  4. Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

    Energy Technology Data Exchange (ETDEWEB)

    Nanda, Dharmin [Department of Neurology, Daniel den Hoed Cancer Centre, University Hospital Rotterdam (Netherlands); Department of Neurosurgery, University Hospital Rotterdam (Netherlands); Jong, Marion de; Bakker, Willem; Bijster, Magda; Cox, Peter [Department of Nuclear Medicine, University Hospital Rotterdam (Netherlands); Vogels, Ronald; Havenga, Menzo [Crucell Holland BV, Leiden (Netherlands); Driesse, Maarten; Avezaat, Cees [Department of Neurosurgery, University Hospital Rotterdam (Netherlands); Morin, Kevin; Naimi, Ebrahim; Knaus, Edward; Wiebe, Leonard [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton (Canada); Smitt, Peter Sillevis [Department of Neurology, Daniel den Hoed Cancer Centre, University Hospital Rotterdam (Netherlands)

    2002-07-01

    Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives.The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy-{beta}-D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy-{beta}-D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of {sup 123}I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with {sup 123}I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). {sup 123}I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy-{beta}-D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with {sup 123}I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after {sup 123}I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with

  5. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    Science.gov (United States)

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

  6. Radiosensitization effect of recombinant adenoviral-mediated PUMA gene on pancreatic carcinoma cells

    International Nuclear Information System (INIS)

    Zhu Dongming; Zhang Kejun; Li Dechun; Zhu Xuefeng; Yang Yong

    2009-01-01

    Objective: To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods: The PANC-1 cells were infected with Ad- PUMA (MOI=10, 50 and 100, respectively) for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 cells were divided into 4 groups: control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups: control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotic index at different time points were recorded in 35 days. Results: The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant (MOI=10, mRNA=0.46± 0.02, protein=0.75± 0.09; MOI=50, mRNA=1.12±0.09, protein=1.01±0.18; MOI=100, mRNA=1.50±0.08, protein= 1.80±0.15; P 3 , (39.5±9.23)mm 3 , (33.6±10.3)mm 3 and (52.0±11.43)mm 3 , respectively, P<0.05]. And the apoptotic index was increased in the same manner (AI=0.43±0.05, 0.29±0.10, 0.24±0.05 and 0.00±0.00, respectively, P<0.05). Conclusions: Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy. (authors)

  7. Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2002-01-01

    Full Text Available Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD along with an optical reporter gene (luciferase. Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging

  8. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Andrew H. Baker

    2010-10-01

    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  9. Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

    1993-01-01

    A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc......-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had...

  10. Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

    Science.gov (United States)

    Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

    2011-01-01

    Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

  11. The oncoprotein gankyrin interacts with RelA and suppresses NF-κB activity

    International Nuclear Information System (INIS)

    Higashitsuji, Hiroaki; Higashitsuji, Hisako; Liu, Yu; Masuda, Tomoko; Fujita, Takanori; Abdel-Aziz, H. Ismail; Kongkham, Supranee; Dawson, Simon; John Mayer, R.; Itoh, Yoshito; Sakurai, Toshiharu; Itoh, Katsuhiko; Fujita, Jun

    2007-01-01

    Gankyrin is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It interacts with multiple proteins and accelerates degradation of tumor suppressors Rb and p53. Since gankyrin consists of 7 ankyrin repeats and is structurally similar to IκBs, we investigated its interaction with NF-κB. We found that gankyrin directly binds to RelA. In HeLa and 293 cells, overexpression of gankyrin suppressed the basal as well as TNFα-induced transcriptional activity of NF-κB, whereas down-regulation of gankyrin increased it. Gankyrin did not affect the NF-κB DNA-binding activity or nuclear translocation of RelA induced by TNFα in these cells. Leptomycin B that inhibits nuclear export of RelA suppressed the NF-κB activity, which was further suppressed by gankyrin. The inhibitory effect of gankyrin was abrogated by nicotinamide as well as down-regulation of SIRT1, a class III histone deacetylase. Thus, gankyrin binds to NF-κB and suppresses its activity at the transcription level by modulating acetylation via SIRT1

  12. The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1).

    Science.gov (United States)

    Hleihel, Rita; Khoshnood, Behzad; Dacklin, Ingrid; Omran, Hayssam; Mouawad, Carine; Dassouki, Zeina; El-Sabban, Marwan; Shirinian, Margret; Grabbe, Caroline; Bazarbachi, Ali

    2018-04-17

    Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic human T-cell lymphotropic virus 1 infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling. Adding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm and major increase in the transcription of the NF-ĸB targets Rantes and interleukin-6. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-κB in flies. These data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL.

  13. The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

    Directory of Open Access Journals (Sweden)

    Sara B Estruch

    Full Text Available Nuclear orphan receptor TLX (NR2E1 functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1, a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9, a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

  14. The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.

    Science.gov (United States)

    Estruch, Sara B; Buzón, Víctor; Carbó, Laia R; Schorova, Lenka; Lüders, Jens; Estébanez-Perpiñá, Eva

    2012-01-01

    Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

  15. The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model

    DEFF Research Database (Denmark)

    Siddique, Hifzur R; Adhami, Vaqar M; Parray, Aijaz

    2013-01-01

    earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4(+/-)) genotype...... (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice......S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We...

  16. Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

    Directory of Open Access Journals (Sweden)

    Juan A Bueren-Calabuig

    2015-06-01

    Full Text Available Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29 peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.

  17. The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal.

    Science.gov (United States)

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Bonilla-Delgado, José; Lagunas-Martínez, Alfredo; Tapia, Jesús Santa-Olalla; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

    2016-12-01

    Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E 2 ) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E 2 on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E 2 in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The effects of combining ionizing radiation and adenoviral p53 therapy in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Li Jianhua; Lax, Stuart A.; Kim, John; Klamut, Henry; Liu Feifei

    1999-01-01

    Purpose: Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region, with a 5-year survival level of approximately 65%. To explore gene therapy as a novel approach which might improve outcome, we have shown previously that introduction of human recombinant wild-type p53 mediated by the adenoviral vector (Ad5CMV-p53) was cytotoxic in two human nasopharyngeal carcinoma (NPC) cell lines (CNE-1 and CNE-2Z). The current work was designed to determine whether this strategy, combined with ionizing radiation (XRT), was more effective than either treatment alone. Methods and Materials: CNE-1, CNE-2Z, and a normal human nasopharyngeal fibroblast strain, KS1, were infected with 2- and 6-plaque-forming units (pfu)/cell of Ad5CMV-p53, respectively. These doses were iso-effective for β-galactosidase activity in the CNE-1 and CNE-2Z cells. XRT was administered 24 h post-infection, and Western blot analyses were conducted for p53, p21 WAF1/CIP1 , bax, and bcl-2 2 days after XRT. Cell survival was assessed using a clonogenic assay. Presence of DNA ladders reflecting apoptosis was detected using DNA agarose gel electrophoresis, and cell cycle was analyzed using flow cytometry. Results: The combination of Ad5CMV-p53 plus XRT (2, 4, and 6 Gy) resulted in an approximately 1-log greater level of cytotoxicity compared to that observed with XRT alone for both NPC cell lines. The two modalities appear to be interacting in a synergistic manner in cancer cells, but not in KS1 fibroblasts. XRT alone stimulated minimal p53 expression in control cells; Ad5CMV-p53 alone induced significant recombinant p53 expression, which was not further enhanced by the addition of XRT. Similar observations were made for p21 WAF1/CIP1 expression. No changes were observed for bax or bcl-2 expression with any of these treatments. Apoptosis was induced following 4 Gy of XRT alone, but was observed after only 2 Gy when combined with Ad5CMV-p53. Cell cycle analysis indicated that Ad5CMV-p53

  19. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

    Science.gov (United States)

    Seandel, Marco; Butler, Jason M; Kobayashi, Hideki; Hooper, Andrea T; White, Ian A; Zhang, Fan; Vertes, Eva L; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V; Hackett, Neil R; Rabbany, Sina; Boyer, Julie L; Rafii, Shahin

    2008-12-09

    Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

  20. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

    Science.gov (United States)

    Seandel, Marco; Butler, Jason M.; Kobayashi, Hideki; Hooper, Andrea T.; White, Ian A.; Zhang, Fan; Vertes, Eva L.; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V.; Hackett, Neil R.; Rabbany, Sina; Boyer, Julie L.; Rafii, Shahin

    2008-01-01

    Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1+ ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1+ ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1+ ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1+ ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1+ ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-κB pathways. Therefore, E4ORF1+ ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis. PMID:19036927

  1. Coating with spermine-pullulan polymer enhances adenoviral transduction of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Wan L

    2016-12-01

    coating could enhance adenoviral transduction of MSCs without detectable cytotoxicity or effects on differentiation. Our results argue in favor of the potentiality of the SP-coated Adv as a prototype vector for efficient and safe transduction of MSCs. Keywords: mesenchymal stem cells, adenovirus vectors, spermine-pullulan, polymer, gene transduction

  2. Toxicity and biodistribution of a first-generation recombinant adenoviral vector, in the presence of hydroxychloroquine, following retroductal delivery to a single rat submandibular gland

    NARCIS (Netherlands)

    Zheng, C.; Voutetakis, A.; Kok, M. R.; Goldsmith, C. M.; Smith, G. B. J.; Elmore, S.; Nyska, A.; Vallant, M.; Irwin, R. D.; Baum, B. J.

    2006-01-01

    We examined the toxicity and biodistribution associated with a single administration of a first-generation, serotype 5, adenoviral vector encoding human growth hormone (hGH; AdCMVhGH) to a single rat submandibular gland in the presence of hydroxychloroquine (HCQ). Previously, we showed that hGH is

  3. Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

    NARCIS (Netherlands)

    Dummer, R; Bergh, J; Karlsson, Y; Horovitz, JA; Mulder, NH; Huinin, DT; Burg, G; Hofbauer, G; Osanto, S

    p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector

  4. Induction of Human Somatostatin Receptor Subtype 2 on Breast Tumors with an Adenoviral Vector for Their Treatment and Detection with a Radiolabeled Peptide

    National Research Council Canada - National Science Library

    Rogers, Buck

    2002-01-01

    .... An adenoviral vector encoding the human somatostatin receptor subtype 2 (AdSSTr2) has been produced. The MDA- MB-468 and BT-474 human breast cancer cells were infected with AdSSTr2 and harvested 48 h later for membrane preparations...

  5. Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo

    NARCIS (Netherlands)

    Holtmaat, Anthony J D G; Hermens, W.T.J.M.C.; Sonnemans, M.A.F.; Giger, Roman J; Van Leeuwen, F W; Kaplitt, M G; Oestreicher, A B; Gispen, Willem Hendrik; Verhaagen, J

    1997-01-01

    B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established

  6. The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

    Directory of Open Access Journals (Sweden)

    Duensing Stefan

    2011-05-01

    Full Text Available Abstract Background Infection with high-risk human papillomaviruses (HPVs such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication. The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4 protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

  7. Production of Human Papilloma Virus Type 16 E6 Oncoprotein as a Recombinant Protein in Eukaryotic Cells

    Science.gov (United States)

    Mirshahabi, H; Soleimanjahi, H; Pourpak, Z; Meshkat, Z; Hassan, ZM

    2012-01-01

    Background Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm. Methods In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/T-E6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5α and transfected into CHO cells 72 hours post-transfection. Results The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody. Conclusion HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies. PMID:25780534

  8. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk [Biomedical Sciences Research Institute, University of Ulster, Coleraine, Co. Derry BT52 1SA (United Kingdom); Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); Patel, Daksha, E-mail: d.patel@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); McCance, Dennis J., E-mail: d.mccance@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom)

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  9. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    International Nuclear Information System (INIS)

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes

  10. Multistage modeling of protein dynamics with monomeric Myc oncoprotein as an example.

    Science.gov (United States)

    Liu, Jiaojiao; Dai, Jin; He, Jianfeng; Niemi, Antti J; Ilieva, Nevena

    2017-03-01

    We propose to combine a mean-field approach with all-atom molecular dynamics (MD) into a multistage algorithm that can model protein folding and dynamics over very long time periods yet with atomic-level precision. As an example, we investigate an isolated monomeric Myc oncoprotein that has been implicated in carcinomas including those in colon, breast, and lungs. Under physiological conditions a monomeric Myc is presumed to be an example of intrinsically disordered proteins that pose a serious challenge to existing modeling techniques. We argue that a room-temperature monomeric Myc is in a dynamical state, it oscillates between different conformations that we identify. For this we adopt the Cα backbone of Myc in a crystallographic heteromer as an initial ansatz for the monomeric structure. We construct a multisoliton of the pertinent Landau free energy to describe the Cα profile with ultrahigh precision. We use Glauber dynamics to resolve how the multisoliton responds to repeated increases and decreases in ambient temperature. We confirm that the initial structure is unstable in isolation. We reveal a highly degenerate ground-state landscape, an attractive set towards which Glauber dynamics converges in the limit of vanishing ambient temperature. We analyze the thermal stability of this Glauber attractor using room-temperature molecular dynamics. We identify and scrutinize a particularly stable subset in which the two helical segments of the original multisoliton align in parallel next to each other. During the MD time evolution of a representative structure from this subset, we observe intermittent quasiparticle oscillations along the C-terminal α helix, some of which resemble a translating Davydov's Amide-I soliton. We propose that the presence of oscillatory motion is in line with the expected intrinsically disordered character of Myc.

  11. A novel basolateral type IV secretion model for the CagA oncoprotein of Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Silja Wessler

    2017-12-01

    Full Text Available Intercellular junctions are crucial structural elements for the formation and maintenance of epithelial barrier functions to control homeostasis or protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of numerous cancers as well as multiple infectious diseases. Many bacterial pathogens harbor type IV secretion systems (T4SSs, which translocate virulence factors into host cells to hijack cellular processes. The pathology of the gastric pathogen and type-I carcinogen Helicobacter pylori strongly depends on a T4SS encoded by the cag pathogenicity island (cagPAI. This T4SS forms a needle-like pilus and its activity is accomplished by the pilus-associated factors CagL, CagI and CagY which target the host integrin-β1 receptor followed by injection of the CagA oncoprotein into non-polarized AGS gastric epithelial cells. The finding of a T4SS receptor, however, suggested the presence of a sophisticated control mechanism for the injection of CagA. In fact, integrins constitute a group of basolateral receptors, which are normally absent at apical surfaces of the polarized epithelium in vivo. Our new results demonstrate that T4SS-pilus formation during H. pylori infection of polarized epithelial cells occurs preferentially at basolateral sites, and not at apical membranes (Tegtmeyer et al., 2017. We propose a stepwise process how H. pylori interacts with components of intercellular tight junctions (TJs and adherens junctions (AJs, followed by contacting integrin-based focal adhesions to disrupt and transform the epithelial cell layer in the human stomach. The possible impact of this novel signaling cascade on pathogenesis during infection is reviewed.

  12. Insight into the tumor suppressor function of CBP through the viral oncoprotein tax.

    Science.gov (United States)

    Van Orden, K; Nyborg, J K

    2000-01-01

    CREB binding protein (CBP) is a cellular coactivator protein that regulates essentially all known pathways of gene expression. The transcriptional coactivator properties of CBP are utilized by at least 25 different transcription factors representing nearly all known classes of DNA binding proteins. Once bound to their target genes, these transcription factors are believed to tether CBP to the promoter, leading to activated transcription. CBP functions to stimulate transcription through direct recruitment of the general transcription machinery as well as acetylation of both histone and transcription factor substrates. Recent observations indicate that a critical dosage of CBP is required for normal development and tumor suppression, and that perturbations in CBP concentrations may disrupt cellular homeostasis. Furthermore, there is accumulating evidence that CBP deregulation plays a direct role in hematopoietic malignancies. However, the molecular events linking CBP deregulation and malignant transformation are unclear. Further insight into the function of CBP, and its role as a tumor suppressor, can be gained through recent studies of the human T-cell leukemia virus, type I (HTLV-I) Tax oncoprotein. Tax is known to utilize CBP to stimulate transcription from the viral promoter. However, recent data suggest that as a consequence of the Tax-CBP interaction, many cellular transcription factor pathways may be deregulated. Tax disruption of CBP function may play a key role in transformation of the HTLV-I-infected cell. Thus, Tax derailment of CBP may lend important information about the tumor suppressor properties of CBP and serve as a model for the role of CBP in hematopoietic malignancies.

  13. Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps.

    Science.gov (United States)

    Leon, B C; Tsigelny, I; Adams, J A

    2001-08-28

    Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881]. A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity. While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower. While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop. Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme. The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected. A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme. While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.

  14. CORRELATION BETWEEN SERUM HER-2 ONCOPROTEIN AND PATIENTS WITH BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    Peng Yuan; Bing-he Xu; Da-tong Chu

    2004-01-01

    Objective To detect serum HER-2 oncoprotein levels in patients with operable and metastatic breast cancers, and to study the correlations between serum HER-2 level and lymph node status as well as other clinical parameters.Methods A total of 120 women were studied consisting of 10 healthy volunteers, 31 benign breast disease, 53 operable breast cancer, and 26 metastatic breast cancer patients. The levels of serum HER-2 were measured using an enzyme-liked immunosorbent assay (ELISA).Results The mean serum HER-2 levels were 9.6 + 1.5 ng/mL in healthy volunteers, 11.9 + 1.6 ng/mL in benign breast disease, 13.2 + 4.2 ng/mL in operable breast cancer, and 30.5 + 30.8 ng/mL in metastatic breast cancer patients. The former is much lower than the latter three (P=0.02, 0.001, 0.03, respectively). If using 15 ng/mL as a normal baseline, elevated serum HER-2 levels were observed in none of the healthy volunteers as well as patients with benign disease, but in 18.9% (10/53)operable breast cancer patients and 61.5% (16/26) metastatic patients. In patients with operable breast cancer, there was a positive correlation between serum concentrations of HER-2 and the size of primary rumor (P < 0.05), whereas there was no correlation between serum concentration and axillary lymph node or estrogen receptor status. In patients with metastatic disease, there was no correlation with site of metastases (P > 0.05).Conclusion Serum HER-2 level was strongly correlated with rumor loads and clinical stages, thus acting as a promising predictor of cancer recurrence in breast cancer patients.

  15. Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity.

    Science.gov (United States)

    Qin, Lingyun; Liu, Huili; Chen, Rong; Zhou, Jingjing; Cheng, Xiyao; Chen, Yao; Huang, Yongqi; Su, Zhengding

    2017-11-07

    The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmX C25-C110/C76-C88 , which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (K d ∼ 0.48 vs K d ∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.

  16. The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿ †

    OpenAIRE

    Spangle, Jennifer M.; Münger, Karl

    2010-01-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y....

  17. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR Results in Severe Inflammation after Adenoviral Gene Therapy.

    Directory of Open Access Journals (Sweden)

    Philipp Christian Seppelt

    Full Text Available Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs. In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1 in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR in order to reduce elastolysis.We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group. Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6 were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1 or β-galactosidase (Ad.β-Gal. As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI, and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM.IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43, but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00. Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001. As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001. However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated

  18. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    International Nuclear Information System (INIS)

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-01

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  19. Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

    Directory of Open Access Journals (Sweden)

    Korson Mark

    2007-04-01

    Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

  20. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Science.gov (United States)

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  1. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  2. The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax

    Science.gov (United States)

    Fryrear, Kimberly A.; Guo, Xin

    2012-01-01

    The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)–modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB–mediated and decreased cAMP Response Element-Binding (CREB)–mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage–induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes. PMID:22106342

  3. Differential gene expression between skin and cervix induced by the E7 oncoprotein in a transgenic mouse model

    Science.gov (United States)

    Ibarra Sierra, E; Díaz Chávez, J; Cortés-Malagón, EM; Uribe-Figueroa, L; Hidalgo-Miranda, A; Lambert, PF; Gariglio, P

    2013-01-01

    HPV16 E7 oncoprotein expression in K14E7 transgenic mice induces cervical cancer after 6 months of treatment with the co-carcinogen 17β-estradiol. In untreated mice, E7 also induces skin tumors late in life albeit at low penetrance. These findings indicate that E7 alters cellular functions in cervix and skin so as to predispose these organs to tumorigenesis. Using microarrays, we determined the global genes expression profile in cervical and skin tissue of young adult K14E7 transgenic mice without estrogen treatment. In these tissues, the E7 oncoprotein altered the transcriptional pattern of genes involved in several biological processes including signal transduction, transport, metabolic process, cell adhesion, apoptosis, cell differentiation, immune response and inflammatory response. Among the E7-dysregulated genes were ones not previously known to be involved in cervical neoplasia including DMBT1, GLI1 and 17βHSD2 in cervix, as well as MMP2, 12, 14, 19 and 27 in skin. PMID:22980503

  4. Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells

    Czech Academy of Sciences Publication Activity Database

    Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Folwarczna, Jitka; Čeřovská, Noemi

    2011-01-01

    Roč. 77, č. 2 (2011), s. 146-152 ISSN 1046-5928 R&D Projects: GA ČR GA521/09/1525 Institutional research plan: CEZ:AV0Z50380511 Keywords : Bacterial expression * Human papillomavirus * Oncoprotein E7 Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.587, year: 2011

  5. The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis.

    Science.gov (United States)

    Spangle, Jennifer M; Münger, Karl

    2010-09-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

  6. Homology Requirements for Efficient, Footprintless Gene Editing at the CFTR Locus in Human iPSCs with Helper-dependent Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Donna J Palmer

    2016-01-01

    Full Text Available Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology, the frequencies of targeted recombinants were 50–64.6% after positive selection for vector integration, and 97.4–100% after negative selection against random integrations. With 14.8 kb, the frequencies were 26.9–57.1% after positive selection and 87.5–100% after negative selection. With 9.6 kb, the frequencies were 21.4 and 75% after positive and negative selection, respectively. With only 5.6 kb, the frequencies were 5.6–16.7% after positive selection and 50% after negative selection, but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore, we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However, low frequencies (≤ 1 × 10−3 necessitated negative selection for piggyBac-excision product isolation.

  7. Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

    Directory of Open Access Journals (Sweden)

    Groitl Peter

    2011-09-01

    Full Text Available Abstract Background Type I interferons (IFNs exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV or HIV. Results Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4+ T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4+ T cell responses were enhanced by IFNα subtypes. Conclusions Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4+ T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines.

  8. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

    Science.gov (United States)

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

    2015-09-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

  9. 5-Fluorouracil-related enhancement of adenoviral infection is Coxsackievirus-adenovirus receptor independent and associated with morphological changes in lipid membranes

    Science.gov (United States)

    Cabrele, Chiara; Vogel, Mandy; Piso, Pompiliu; Rentsch, Markus; Schröder, Josef; Jauch, Karl W; Schlitt, Hans J; Beham, Alexander

    2006-01-01

    AIM: To evaluate the mechanism underlying the effects of 5-Fluorouracil (5-FU) on adenoviral infection. METHODS: Low and high Coxsackievirus-Adenovirus Receptor (CAR) expressing human colon carcinoma cell lines were treated with 5-FU and two E1-deleted adenoviral constructs, one transferring GFP (Ad/CMV-GFP) the other bax (Ad/CEA-bax). The number of infected cells were monitored by GFP expression. To evaluate the effects of 5-FU in a receptor free system, Ad/GFP were encapsulated in liposomes and treated with 5-FU. Ad/GFP release was estimated with PCR and infection of 293 cells with the supernatant. Electron microscopy of the Ad5-GFP-liposome complex was made to investigate morphological changes of the liposomes after 5-FU. RESULTS: Infection rates of all cell lines increased from 50% to 98% with emerging 5-FU concentrations. The enhanced viral uptake was independent of the CAR expression. Additionally, 5-FU treated liposomes released 2-2.5 times more adenoviruses. Furthermore, 5-FU-treated liposomes appeared irregular and porous-like. CONCLUSION: adenoviral uptake is enhanced in the presence of 5-FU irrespective of CAR and is associated with morphological changes in membranes making the combination of both a promising option in gene therapy. PMID:16937527

  10. MUC1-C Oncoprotein Integrates a Program of EMT, Epigenetic Reprogramming and Immune Evasion in Human Carcinomas.

    Science.gov (United States)

    Rajabi, Hasan; Kufe, Donald

    2017-08-01

    The MUC1 gene evolved in mammalian species to provide protection of epithelia. The transmembrane MUC1 C-terminal subunit (MUC1-C) signals stress to the interior of the epithelial cell and, when overexpressed as in most carcinomas, functions as an oncoprotein. MUC1-C induces the epithelial-mesenchymal transition (EMT) by activating the inflammatory NF-κB p65 pathway and, in turn, the EMT-transcriptional repressor ZEB1. Emerging evidence has indicated that MUC1-C drives a program integrating the induction of EMT with activation of stem cell traits, epigenetic reprogramming and immune evasion. This mini-review focuses on the potential importance of this MUC1-C program in cancer progression. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yueyang [Division of Infectious Diseases, Brigham and Women' s Hospital and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115 (United States); Munger, Karl, E-mail: kmunger@rics.bwh.harvard.edu [Division of Infectious Diseases, Brigham and Women' s Hospital and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115 (United States)

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

  12. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    Directory of Open Access Journals (Sweden)

    Vijayendra Dasari

    2016-01-01

    Full Text Available Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients.

  13. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    Science.gov (United States)

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  14. Efficient expression of Human papillomavirus 16 E7 oncoprotein fused to C-terminus of Tobacco mosaic virus (TMV) coat protein using molecular chaperones in Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Folwarczna, Jitka; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Čeřovská, Noemi

    2012-01-01

    Roč. 85, č. 1 (2012), s. 152-157 ISSN 1046-5928 R&D Projects: GA ČR GA521/09/1525; GA ČR GAP501/12/1761 Institutional research plan: CEZ:AV0Z50380511 Keywords : Bacterial expression * Human papillomavirus * E7 oncoprotein Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.429, year: 2012

  15. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona, E-mail: moroianu@bc.edu

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  16. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway.

    Science.gov (United States)

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP-8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), 76IRTFQELLF84, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells

    International Nuclear Information System (INIS)

    Accardi, Luisa; Tommasino, Massimo; Banks, Lawrence; Chirullo, Barbara; Giorgi, Colomba; Donà, Maria Gabriella; Mileo, Anna M; Paggi, Marco G; Federico, Antonio; Torreri, Paola; Petrucci, Tamara C; Accardi, Rosita; Pim, David

    2011-01-01

    'High risk' Human Papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The 'intracellular antibody' technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to 'knock out' the function of specific proteins. In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the 'high-risk' HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™technology and Surface Plasmon Resonance. The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic

  18. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: kerstin.abshagen@uni-rostock.de [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  19. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    International Nuclear Information System (INIS)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-01-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells

  20. Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction

    Science.gov (United States)

    Li, Longhu; Haider, Husnain Kh.; Wang, Linlin; Lu, Gang

    2012-01-01

    We previously showed that treatment with tadalafil, a long-acting phosphodiesterase-5a (PDE5a) inhibitor, effectively prevented adverse left ventricular (LV) remodeling of the infarcted heart. We hypothesized that short-hairpin RNA (shRNA) therapy targeting PDE5a would simulate the effects of pharmacological intervention for treatment of postinfarction LV remodeling and dysfunction. Experimental model of myocardial infarction was developed in female mice by permanent ligation of left coronary artery. Immediately after that, an adenoviral vector encoding for shRNA sequence targeting PDE5a (Ad-shPDE5a) was injected intramyocardially, which specifically inhibited PDE5a in the heart. Four weeks later, Ad-shPDE5a treated mice showed significant mitigation of the left ventricle (LV) dilatation and dysfunction as indicated by smaller LV cavity and more preserved ejection fraction and fractional shortening. Infarction size and fibrosis were significantly reduced in Ad-shPDE5a-treated mice. Additionally, more salvaged cardiomyocytes, significantly reduced collagen contents, and higher blood vessel density were observed in Ad-shPDE5a-treated mice. The cytoprotective effects of Ad-shPDE5a were demonstrated in vitro in Ad-shPDE5a transfected cardiomyocytes cultured under oxygen glucose deprivation. Among downstream mediators of PDE5a signaling, cyclic GMP (cGMP) and cGMP-dependent protein kinase G (PKG) were activated with concomitant reduction in caspase-3 activity. However, no significant change in PKA and cAMP activities were observed in Ad-shPDE5a-treated hearts. Inhibition with shRNA improved cardiac remodeling and dysfunction by reducing infarction size and cardiac fibrosis and increased cGMP and PKG activity. These findings suggest that PDE5 inhibition with Ad-shPDE5a is a novel approach for treatment of myocardial infarction. PMID:22447941

  1. Lysyl Oxidase-Like 1 Protein Deficiency Protects Mice from Adenoviral Transforming Growth Factor-β1-induced Pulmonary Fibrosis.

    Science.gov (United States)

    Bellaye, Pierre-Simon; Shimbori, Chiko; Upagupta, Chandak; Sato, Seidai; Shi, Wei; Gauldie, Jack; Ask, Kjetil; Kolb, Martin

    2018-04-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The abnormal ECM deposition slowly overtakes normal lung tissue, disturbing gas exchange and leading to respiratory failure and death. ECM cross-linking and subsequent stiffening is thought to be a major contributor of disease progression and also promotes the activation of transforming growth factor (TGF)-β1, one of the main profibrotic growth factors. Lysyl oxidase-like (LOXL) 1 belongs to the cross-linking enzyme family and has been shown to be up-regulated in active fibrotic regions of bleomycin-treated mice and patients with IPF. We demonstrate in this study that LOXL1-deficient mice are protected from experimental lung fibrosis induced by overexpression of TGF-β1 using adenoviral (Ad) gene transfer (AdTGF-β1). The lack of LOXL1 prevented accumulation of insoluble cross-linked collagen in the lungs, and therefore limited lung stiffness after AdTGF-β1. In addition, we applied mechanical stretch to lung slices from LOXL1 +/+ and LOXL1 -/- mice treated with AdTGF-β1. Lung stiffness (Young's modulus) of LOXL1 -/- lung slices was significantly lower compared with LOXL1 +/+ lung slices. Moreover, the release of activated TGF-β1 after mechanical stretch was significantly lower in LOXL1 -/- mice compared with LOXL1 +/+ mice after AdTGF-β1. These data support the concept that cross-linking enzyme inhibition represents an interesting therapeutic target for drug development in IPF.

  2. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71.

    Science.gov (United States)

    Zhang, Chao; Yang, Yong; Chi, Yudan; Yin, Jieyun; Yan, Lijun; Ku, Zhiqiang; Liu, Qingwei; Huang, Zhong; Zhou, Dongming

    2015-09-22

    Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexon-modified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    International Nuclear Information System (INIS)

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-01-01

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  4. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Su, Xiong [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Liu, Jialiu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Sundaresan, Sinju [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Stahl, Philip D., E-mail: pstahl@wustl.edu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  5. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  6. H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

    Science.gov (United States)

    Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

    2017-02-01

    Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

  7. Interplay Between Oncoproteins and Antioxidant Enzymes in Esophageal Carcinoma Treated Without and With Chemoradiotherapy: A Prospective Study

    International Nuclear Information System (INIS)

    Kaur, Tranum; Gupta, Rajesh; Vaiphei, Kim; Kapoor, Rakesh; Gupta, N.M.; Khanduja, K.L.

    2008-01-01

    Purpose: To analyze p53, bcl-2, c-myc, and cyclooxygenase-2 protein expression changes and examine their relationship with various antioxidant enzymes in esophageal carcinoma patients. Methods and Materials: Patients in Group 1 underwent transhiatal esophagectomy and those in Group 2 were administered chemoradiotherapy followed by surgery after 4 weeks of neoadjuvant therapy. Results: The relationship analysis among the various protein markers and antioxidant enzymes showed an inverse correlation between bcl-2 and superoxide dismutase/catalase in tumor tissues, irrespective of the treatment arm followed. An important positive association was observed between bcl-2 and reduced glutathione levels in the tumor tissue of patients receiving neoadjuvant therapy. Another apoptosis-modulating marker, c-myc, in the tumor tissue of Group 2 patients showed similar pattern levels (high and low) as that of superoxide dismutase/catalase. The association of cyclooxygenase-2 and p53 with various antioxidant enzymes showed a significant positive correlation between cyclooxygenase-2 expression and catalase activity and an inverse trend between p53 expression and superoxide dismutase and catalase activity in the tumor tissue of patients given neoadjuvant therapy. In addition, patients with overexpressed p53 protein levels had lower glutathione peroxidase enzyme levels and vice versa in the tumor tissue of patients who had undergone surgery as their main mode of treatment. Conclusion: The results of this study broaden the insight into the relationships shared among oncoproteins and the antioxidant defense system, and this could be helpful in the clinical management of esophageal carcinoma

  8. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    International Nuclear Information System (INIS)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli; Zhang, Xiaodong; Ye, Lihong

    2013-01-01

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells

  9. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    Science.gov (United States)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Ex vivo adenoviral vector-mediated neurotrophin gene transfer to olfactory ensheathing glia : effects on rubrospinal tract regeneration, lesion size, and functional recovery after implantation in the injured rat spinal cord

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Plant, Giles W; Hamers, Frank P T; Wortel, Joke; Blits, Bas; Dijkhuizen, Paul A; Gispen, Willem Hendrik; Boer, Gerard J; Verhaagen, J.

    2003-01-01

    The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived

  11. HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

    Science.gov (United States)

    Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

    2008-01-01

    Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

  12. Alternative Splicing and Caspase-Mediated Cleavage Generate Antagonistic Variants of the Stress Oncoprotein LEDGF/p75

    Science.gov (United States)

    Brown-Bryan, Terry A.; Leoh, Lai S.; Ganapathy, Vidya; Pacheco, Fabio J.; Mediavilla-Varela, Melanie; Filippova, Maria; Linkhart, Thomas A.; Gijsbers, Rik; Debyser, Zeger; Casiano, Carlos A.

    2009-01-01

    There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium–derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress–induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH2-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies. PMID:18708362

  13. DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

    International Nuclear Information System (INIS)

    Lutwyche, Jodi K.; Keough, Rebecca A.; Hunter, Julie; Coles, Leeanne S.; Gonda, Thomas J.

    2006-01-01

    Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor

  14. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    Science.gov (United States)

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  15. Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt.

    Science.gov (United States)

    Deng, Gang; Huang, Xiang-Jun; Luo, Hong-Wu; Huang, Fei-Zhou; Liu, Xun-Yang; Wang, Yong-Heng

    2013-01-01

    To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 10(11) vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups. The results of HE and

  16. Aspirin therapy reduces the ability of platelets to promote colon and pancreatic cancer cell proliferation: Implications for the oncoprotein c-MYC

    Science.gov (United States)

    Sylman, Joanna L.; Ngo, Anh T. P.; Pang, Jiaqing; Sears, Rosalie C.; Williams, Craig D.; McCarty, Owen J. T.

    2017-01-01

    Aspirin, an anti-inflammatory and antithrombotic drug, has become the focus of intense research as a potential anticancer agent owing to its ability to reduce tumor proliferation in vitro and to prevent tumorigenesis in patients. Studies have found an anticancer effect of aspirin when used in low, antiplatelet doses. However, the mechanisms through which low-dose aspirin works are poorly understood. In this study, we aimed to determine the effect of aspirin on the cross talk between platelets and cancer cells. For our study, we used two colon cancer cell lines isolated from the same donor but characterized by different metastatic potential, SW480 (nonmetastatic) and SW620 (metastatic) cancer cells, and a pancreatic cancer cell line, PANC-1 (nonmetastatic). We found that SW480 and PANC-1 cancer cell proliferation was potentiated by human platelets in a manner dependent on the upregulation and activation of the oncoprotein c-MYC. The ability of platelets to upregulate c-MYC and cancer cell proliferation was reversed by an antiplatelet concentration of aspirin. In conclusion, we show for the first time that inhibition of platelets by aspirin can affect their ability to induce cancer cell proliferation through the modulation of the c-MYC oncoprotein. PMID:27903583

  17. Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

    Science.gov (United States)

    Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

    2014-01-01

    Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

  18. Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity.

    Science.gov (United States)

    Li, Junan; Li, Hongyuan; Tsai, Ming-Daw

    2003-06-10

    The involvement of Tax oncoprotein in the INK4-CDK4/6-Rb pathway has been regarded as a key factor for immortalization and transformation of human T-cell leukemia virus 1 (HTLV-1) infected cells. In both p16 -/- and +/+ cells, expression of Tax has been correlated with an increase in CDK4 activity, which subsequently increases the phosphorylation of Rb and drives the infected cells into cell cycle progression. In relation to these effects, Tax has been shown to interact with two components of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s). While Tax competes with CDK4 for p16 binding, thus suppressing p16 inhibition of CDK4, Tax also binds to cyclin D(s) with concomitant increases in both CDK4 activity and the phosphorylation of cyclin D(s). Here we show that both Tax and residues 1-40 of the N-terminus of Tax, Tax40N, bind to and activate CDK4 in vitro. In the presence of INK4 proteins, binding of Tax and Tax40N to CDK4 counteracts against the inhibition of p16 and p18 and acts as the major path to regulate Tax-mediated activation of CDK4. We also report that Tax40N retains the transactivation ability. These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.

  19. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load

    DEFF Research Database (Denmark)

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P

    2009-01-01

    -68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential...... of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus......-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral...

  20. Comparison of [{sup 18}F]FHBG and [{sup 14}C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung-Jun; Iyer, Meera [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Gambhir, Sanjiv S. [The Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, B3-399A BRI 700 Westwood Plaza, CA 90095-1770, Los Angeles (United States); Department of Radiology, Bio-X Program, Stanford University, Stanford, California (United States)

    2003-11-01

    Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells - stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [{sup 18}F]FHBG, [{sup 3}H]PCV, and [{sup 14}C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [{sup 14}C]FIAU accumulation was greater than that of [{sup 3}H]PCV and [{sup 18}F]FHBG in control cells and in HSV1-tk stably transfected cells (P<0.001). After infection of C6 cells with AdCMV-HSV1-tk (1.5 x 10{sup 8} pfu), [{sup 18}F]FHBG and [{sup 3}H]PCV accumulation was significantly greater than that of [{sup 14}C]FIAU (P<0.01). [{sup 18}F]FHBG and [{sup 3}H]PCV accumulated to a significantly greater extent than [{sup 14}C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P<0.001). Results from the nude mice supported the results in cell culture. [{sup 14}C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [{sup 18}F]FHBG (P<0.05); however, compared with [{sup 14}C]FIAU, [{sup 18}F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [{sup 18}F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A

  1. An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

    Directory of Open Access Journals (Sweden)

    Chitose Kurihara

    2016-12-01

    Full Text Available The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL. This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre. Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF into induced-hepatocyte-like cells (iHep following several rounds of infection, demonstrating the efficacy of this new system.

  2. Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation

    International Nuclear Information System (INIS)

    DeWeese, Theodore L.; Walsh, Jonathan C.; Dillehay, Larry E.; Kessis, Theodore D.; Hedrick, Lora; Cho, Kathleen R.; Nelson, William G.

    1997-01-01

    Purpose: Low-dose-rate radiation therapy has been widely used in the treatment of urogenital malignancies. When continuously exposed to low-dose-rate ionizing radiation, target cancer cells typically exhibit abnormalities in replicative cell-cycle progression. Cancer cells that arrest in the G2 phase of the cell cycle when irradiated may become exquisitely sensitive to killing by further low-dose-rate radiation treatment. Oncogenic human papillomaviruses (HPVs), which play a major role in the pathogenesis of uterine cervix cancers and other urogenital cancers, encode E6 and E7 transforming proteins known to abrogate a p53-dependent G1 cell-cycle checkpoint activated by conventional acute-dose radiation exposure. This study examined whether expression of HPV E6 and E7 oncoproteins by cancer cells alters the cell-cycle redistribution patterns accompanying low-dose-rate radiation treatment, and whether such alterations in cell-cycle redistribution affect cancer cell killing. Methods and Materials: RKO carcinoma cells, which contain wild-type P53 alleles, and RKO cell sublines genetically engineered to express HPV E6 and E7 oncoproteins, were treated with low-dose-rate (0.25-Gy/h) radiation and then assessed for p53 and p21WAF1/CIP1 polypeptide induction by immunoblot analysis, for cell-cycle redistribution by flow cytometry, and for cytotoxicity by clonogenic survival assay. Results: Low-dose-rate radiation of RKO carcinoma cells triggered p53 polypeptide elevations, p21WAF1/CIP1 induction, and arrest in the G1 and G2 phases of the cell cycle. In contrast, RKO cells expressing E6 and E7 transforming proteins from high-risk oncogenic HPVs (HPV 16) arrested in G2, but failed to arrest in G1, when treated with low-dose-rate ionizing radiation. Abrogation of the G1 cell-cycle checkpoint activated by low-dose-rate radiation exposure appeared to be a characteristic feature of transforming proteins from high-risk oncogenic HPVs: RKO cells expressing E6 from a low

  3. Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

    Directory of Open Access Journals (Sweden)

    Gauldie Jack

    2010-12-01

    Full Text Available Abstract Background Micro-computed tomography (micro-CT is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time. Methods Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1. Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis. Results We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed. Conclusions Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.

  4. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

    International Nuclear Information System (INIS)

    Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

    2004-01-01

    Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

  5. Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

    Directory of Open Access Journals (Sweden)

    Teschemacher Anja G

    2009-03-01

    Full Text Available Abstract Background 5-hydroxytryptamine (5 HT, serotonin is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain. Results We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2 gene which selectively (97% co-localisation with TPH-2 target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules. Conclusion For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this

  6. Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

    Science.gov (United States)

    Groussaud, Damien; Khair, Mostafa; Tollenaere, Armelle I; Waast, Laetitia; Kuo, Mei-Shiue; Mangeney, Marianne; Martella, Christophe; Fardini, Yann; Coste, Solène; Souidi, Mouloud; Benit, Laurence; Pique, Claudine; Issad, Tarik

    2017-07-01

    The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway

  7. c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin

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    Alejandro J. Moyano

    2018-05-01

    Full Text Available c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant

  8. UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins.

    Science.gov (United States)

    Pacini, Laura; Ceraolo, Maria Grazia; Venuti, Assunta; Melita, Giusi; Hasan, Uzma A; Accardi, Rosita; Tommasino, Massimo

    2017-10-01

    Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis. IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53

  9. Priming of CD8 T Cells by Adenoviral Vectors Is Critically Dependent on B7 and Dendritic Cells but Only Partially Dependent on CD28 Ligation on CD8 T Cells

    DEFF Research Database (Denmark)

    Nielsen, Karen N; Steffensen, Maria A; Christensen, Jan P

    2014-01-01

    expression resulted in a delayed primary response, whereas memory CD8 T cells generated in CD28-deficient mice appeared almost normal in terms of both phenotype and effector cytokine profile, but they exhibited a significantly reduced proliferative capacity upon secondary challenge while retaining immediate...... in vivo effector capabilities: in vivo cytotoxicity and short-term in vivo protective capacity. Overall, our data point to an absolute requirement for professional APCs and the expression of the costimulatory molecules CD80/86 for efficient CD8 T cell priming by adenoviral vectors. Additionally, our......Adenoviral vectors have long been forerunners in the development of effective CD8 T cell-based vaccines; therefore, it is imperative that we understand the factors controlling the induction of robust and long-lasting transgene-specific immune responses by these vectors. In this study, we...

  10. Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

    Science.gov (United States)

    Zhao, Huzi; Zhang, Lina; Zhang, Yongchen; Zhao, Lei; Wan, Qing; Wang, Bei; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

    2017-05-30

    The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

  11. Optimization and physicochemical characterization of a cationic lipid-phosphatidylcholine mixed emulsion formulated as a highly efficient vehicle that facilitates adenoviral gene transfer

    Directory of Open Access Journals (Sweden)

    Kim SY

    2017-10-01

    Full Text Available Soo-Yeon Kim,1,2 Sang-Jin Lee,2 Jin-Ki Kim,3 Han-Gon Choi,3 Soo-Jeong Lim1 1Department of Bioscience and Bioengineering, Sejong University, Seoul, Kwangjin-gu, Seoul, 2Immunotherapeutics Branch, Research Institute, National Cancer Center, Ilsandong-gu, Goyang-si, Gyeonggi-do, 3College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, Sangnok-gu, Ansan, Republic of Korea Abstract: Cationic lipid-based nanoparticles enhance viral gene transfer by forming electrostatic complexes with adenoviral vectors. We recently demonstrated the superior complexation capabilities of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP emulsion compared with a liposomal counterpart but the cytotoxicity of DOTAP emulsions remained a challenge. The present study is aimed at formulating an emulsion capable of acting as a highly effective viral gene transfer vehicle with reduced cytotoxicity and to physicochemically characterize the structures of virus-emulsion complexes in comparison with virus–liposome complexes when the only difference between emulsions and liposomes was the presence or absence of inner oil core. The emulsion formulation was performed by 1 reducing the content of DOTAP while increasing the content of zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, and 2 optimizing the oil content. The complexation capability of formulated DOTAP:DMPC mixed emulsions was similar to those of emulsions containing DOTAP alone while displaying significantly lower cytotoxicity. The complexation capabilities of the DOTAP:DMPC mixed emulsion were serum-compatible and were monitored in a variety of cell types, whereas its liposomal counterpart was totally ineffective. Characterization by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and dynamic light scattering studies indicated that the optimized emulsions spontaneously surrounded the virus particles to generate emulsions that

  12. Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

    Directory of Open Access Journals (Sweden)

    Angela Cassese

    Full Text Available Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15 causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1. Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4 restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15 by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

  13. Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice - role of regulatory T cells.

    Science.gov (United States)

    Singpiel, Alena; Kramer, Julia; Maus, Regina; Stolper, Jennifer; Bittersohl, Lara Friederike; Gauldie, Jack; Kolb, Martin; Welte, Tobias; Sparwasser, Tim; Maus, Ulrich A

    2018-03-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγ pos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    Directory of Open Access Journals (Sweden)

    Daria A Burmistrova

    Full Text Available Developing pathogen-specific recombinant antibody fragments (especially nanobodies is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh, for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection.

  15. Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma.

    Science.gov (United States)

    Jin, D Y; Giordano, V; Kibler, K V; Nakano, H; Jeang, K T

    1999-06-18

    Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-kappaB remain incompletely understood. Although others have described an interaction between Tax and a holo-IkappaB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-alpha nor IKK-beta but instead complexes directly with IKK-gamma, a newly characterized component of the IKK complex. This direct interaction with IKK-gamma correlates with Tax-induced IkappaB-alpha phosphorylation and NF-kappaB activation. Thus, our findings establish IKK-gamma as a key molecule for adapting an oncoprotein-specific signaling to IKK-alpha and IKK-beta.

  16. Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

    Science.gov (United States)

    Popławski, Piotr; Wiśniewski, Jacek R; Rijntjes, Eddy; Richards, Keith; Rybicka, Beata; Köhrle, Josef; Piekiełko-Witkowska, Agnieszka

    2017-01-01

    Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3'-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes

  17. Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

    Directory of Open Access Journals (Sweden)

    Piotr Popławski

    Full Text Available Type 1 iodothyronine deiodinase (DIO1 contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4 yielding of 3,5,3'-triiodothyronine (T3, a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2 that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2, enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2, sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10. DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression

  18. Post-spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG.

    Science.gov (United States)

    Povysheva, Tatyana; Shmarov, Maksim; Logunov, Denis; Naroditsky, Boris; Shulman, Ilya; Ogurcov, Sergey; Kolesnikov, Pavel; Islamov, Rustem; Chelyshev, Yuri

    2017-07-01

    OBJECTIVE The most actively explored therapeutic strategy for overcoming spinal cord injury (SCI) is the delivery of genes encoding molecules that stimulate regeneration. In a mouse model of amyotrophic lateral sclerosis and in preliminary clinical trials in patients with amyotrophic lateral sclerosis, the combined administration of recombinant adenoviral vectors (Ad5-VEGF+Ad5-ANG) encoding the neurotrophic/angiogenic factors vascular endothelial growth factor ( VEGF) and angiogenin ( ANG) was found to slow the development of neurological deficits. These results suggest that there may be positive effects of this combination of genes in posttraumatic spinal cord regeneration. The objective of the present study was to determine the effects of Ad5-VEGF+Ad5-ANG combination therapy on motor function recovery and reactivity of astrocytes in a rat model of SCI. METHODS Spinal cord injury was induced in adult Wistar rats by the weight-drop method. Rats (n = 51) were divided into 2 groups: the experimental group (Ad5-VEGF+Ad5-ANG) and the control group (Ad5-GFP [green fluorescent protein]). Recovery of motor function was assessed using the Basso, Beattie, and Bresnahan scale. The duration and intensity of infectivity and gene expression from the injected vectors were assessed by immunofluorescent detection of GFP. Reactivity of glial cells was assessed by changes in the number of immunopositive cells expressing glial fibrillary acidic protein (GFAP), S100β, aquaporin 4 (AQP4), oligodendrocyte transcription factor 2, and chondroitin sulfate proteoglycan 4. The level of S100β mRNA expression in the spinal cord was estimated by real-time polymerase chain reaction. RESULTS Partial recovery of motor function was observed 30 days after surgery in both groups. However, Basso, Beattie, and Bresnahan scores were 35.9% higher in the Ad5-VEGF+Ad5-ANG group compared with the control group. Specific GFP signal was observed at distances of up to 5 mm in the rostral and caudal

  19. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load.

    Science.gov (United States)

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P; Holst, Peter J

    2009-11-12

    Gammaherpesviruses establish life-long latent infections in their hosts. If the host becomes immunosuppressed, these viruses may reactivate and cause severe disease, and even in immunocompetent individuals the gammaherpesviruses are presumed to have an oncogenic potential. Murine gammaherpesvirus-68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral vectors encoding both M2 and M3. Additionally we show that M3 immunization prevented the usual development of virus-induced splenomegaly at 2-3 weeks post-infection. This is the first time that immunization with a non-replicating vaccine has lead to a significantly reduced viral load at time points beyond 14 days post-infection, and thus demonstrates that a non-replicating vaccine may successfully be employed to reduce the viral burden during chronic gammaherpesvirus infection.

  20. INGN 201: Ad-p53, Ad5CMV-p53, Adenoviral p53, INGN 101, p53 gene therapy--Introgen, RPR/INGN 201.

    Science.gov (United States)

    2003-01-01

    Introgen's adenoviral p53 gene therapy [INGN 201, ADVEXIN] is in clinical development for the treatment of various cancers. The p53 tumour suppressor gene is deleted or mutated in many tumour cells and is one of the most frequently mutated genes in human tumours. INGN 201 has been shown to kill cancer cells directly. In August 2002, Introgen announced plans to file an application for INGN 201 with the European Agency for the Evaluation of Medicinal Products (EMEA) for the treatment of head and neck cancer; the European filing will be submitted simultaneously with the previously scheduled (planned for 2004) submission of a Biologics License Application (BLA) for ADVEXIN to the US FDA. On 20 February 2003, INGN 201 received orphan drug designation from the US FDA for head and neck cancer. INGN 201 is available for licensing although Introgen favours retaining partial or full rights to the therapy in the US. Introgen Therapeutics and its collaborative partner for the p53 programme, Aventis Gencell, have been developing p53 gene therapy products. The agreement was originally signed by Rhône-Poulenc Rorer's Gencell division, which became Aventis Gencell after Rhône-Poulenc Rorer merged with Hoechst Marion Roussel to form Aventis Pharma. According to the original agreement, Introgen was responsible for phase I and preclinical development in North America, while Aventis Gencell was responsible for clinical trials conducted in Europe and for clinical trials in North America beyond phase I. In April 2001, Aventis Gencell and Introgen restructured their existing collaboration agreement for p53 gene therapy products. Aventis Gencell indicated that p53 research had suffered from internal competition for resources and was pulling back from its development agreement with Introgen for p53 gene therapy products. Introgen will assume responsibility for worldwide development of all p53 programmes and will obtain exclusive worldwide commercial rights to p53-based gene therapy

  1. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

    Directory of Open Access Journals (Sweden)

    Alexander J Neumann

    Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

  2. Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

    Directory of Open Access Journals (Sweden)

    Pavlo L Kovalenko

    Full Text Available Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3 is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3 or silencing RNA for Slfn3 (siSlfn3 was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI, Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.

  3. Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells

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    Mohamed A. El-Mogy

    2017-06-01

    Full Text Available The adenovirus major late promoter (MLP and its translational regulator – the tripartite leader (TPL sequence – can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds than translation (11 folds. Even with adenovirus infection to provide additional elements (in trans, CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.

  4. Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model.

    Science.gov (United States)

    Müller, Jakob; Thirion, Christian; Pfaffl, Michael W

    2011-01-15

    Recombinant viral vectors are widespread tools for transfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR) based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a current flow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell-cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approach shows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p<0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes.

    Science.gov (United States)

    Hurtado, Cecilia; Ander, Bradley P; Maddaford, Thane G; Lukas, Anton; Hryshko, Larry V; Pierce, Grant N

    2005-04-01

    The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.

  6. Experimental study on the effects of recombinant adenoviral-mediated mI{kappa}B{alpha} gene combined with irradiation on the treatment of hepatocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kejun, Zhang; Dechun, Li; Dongming, Zhu [The First Affiliated Hospital to Suzhou Univ., Suzhou (China); Caixia, Song

    2007-10-15

    Objective: To explore the effect of recombinant adenovirus vector mediated mutant I{kappa}B{alpha} (mI{kappa}B{alpha}) combined with radiation on the hepatocarcinoma. Methods: Limited dilution method was used to test the virus titer in 293 cells. The HCC9204 cells were infected with MOI 10,20,30 and 50 for 48 h, respectively. The expression of p65 and mI{kappa}B{alpha} protein was analyzed by Western blot. Transfected HCC9204 cells and controls were treated with 4 Gy {gamma} rays. The inhibition rate of HCC9204 cells was examined by MTT. Rat models of HCC9204 was constructed. AdmI{kappa}B{alpha} plasmids were injected into tumor tissue and the tumors were administered with 6 Gy {gamma} irradiation 48 hours later. Tumor growth at different time points was recorded during 28 days. Results: The titer of AdmI{kappa}B{alpha} is 1.252 x 10{sup 9} pfu/ml. The expression of mI{kappa}B{alpha} protein was increased with titer of AdmI{kappa}B{alpha}, and p65 protein began to decrease when MOI was 10, and reached the lowest when MOI was 50, they were all dose-dependent. The proliferation of HCC9204 cell lines were suppressed, as was more significant combined with radiation, and the effect was in a viral dose-dependent manner. From days 7 to 28 after AdmI{kappa}B{alpha} gene and radiotherapy, the tumor growth was significantly slower than after irradiation or gene therapy alone. Conclusions: Recombinant adenoviral-mediated mI{kappa}B{alpha} gene, combined with irradiation, can increase the cell-killing effect. It is better than that of either one alone. (authors)

  7. Experimental study on the effects of recombinant adenoviral-mediated mIκBα gene combined with irradiation on the treatment of hepatocarcinoma

    International Nuclear Information System (INIS)

    Zhang Kejun; Li Dechun; Zhu Dongming; Song Caixia

    2007-01-01

    Objective: To explore the effect of recombinant adenovirus vector mediated mutant IκBα (mIκBα) combined with radiation on the hepatocarcinoma. Methods: Limited dilution method was used to test the virus titer in 293 cells. The HCC9204 cells were infected with MOI 10,20,30 and 50 for 48 h, respectively. The expression of p65 and mIκBα protein was analyzed by Western blot. Transfected HCC9204 cells and controls were treated with 4 Gy γ rays. The inhibition rate of HCC9204 cells was examined by MTT. Rat models of HCC9204 was constructed. AdmIκBα plasmids were injected into tumor tissue and the tumors were administered with 6 Gy γ irradiation 48 hours later. Tumor growth at different time points was recorded during 28 days. Results: The titer of AdmIκBΑ is 1.252 x 10 9 pfu/ml. The expression of mIκBα protein was increased with titer of AdmIκBα, and p65 protein began to decrease when MOI was 10, and reached the lowest when MOI was 50, they were all dose-dependent. The proliferation of HCC9204 cell lines were suppressed, as was more significant combined with radiation, and the effect was in a viral dose-dependent manner. From days 7 to 28 after AdmIκBα gene and radiotherapy, the tumor growth was significantly slower than after irradiation or gene therapy alone. Conclusions: Recombinant adenoviral-mediated mIκBα gene, combined with irradiation, can increase the cell-killing effect. It is better than that of either one alone. (authors)

  8. PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein augments the transforming activity in a rat fibroblast cell line

    International Nuclear Information System (INIS)

    Hirata, Akira; Higuchi, Masaya; Niinuma, Akiko; Ohashi, Minako; Fukushi, Masaya; Oie, Masayasu; Akiyama, Tetsu; Tanaka, Yuetsu; Gejyo, Fumitake; Fujii, Masahiro

    2004-01-01

    While human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL), HTLV-2 has not been reported to be associated with such malignant leukemias. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) to form multiple large colonies in soft agar, and this activity is much greater than that of HTLV-2 Tax2. We have demonstrated here that the increased number of transformed colonies induced by Tax1 relative to Tax2 was mediated by a PDZ domain-binding motif (PBM) in Tax1, which is absent in Tax2. Tax1 PBM mediated the interaction of Tax1 with the discs large (Dlg) tumor suppressor containing PDZ domains, and the interaction correlated well with the transforming activities of Tax1 and the mutants. Through this interaction, Tax1 altered the subcellular localization of Dlg from the detergent-soluble to the detergent-insoluble fraction in a fibroblast cell line as well as in HTLV-1-infected T-cell lines. These results suggest that the interaction of Tax1 with PDZ domain protein(s) is critically involved in the transforming activity of Tax1, the activity of which may be a crucial factor in malignant transformation of HTLV-1-infected cells in vivo

  9. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax.

    Science.gov (United States)

    Mann, Melanie C; Strobel, Sarah; Fleckenstein, Bernhard; Kress, Andrea K

    2014-09-01

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    Energy Technology Data Exchange (ETDEWEB)

    Mann, Melanie C., E-mail: melanie.mann@viro.med.uni-erlangen.de; Strobel, Sarah, E-mail: sarah.strobel@viro.med.uni-erlangen.de; Fleckenstein, Bernhard, E-mail: bernhard.fleckenstein@viro.med.uni-erlangen.de; Kress, Andrea K., E-mail: andrea.kress@viro.med.uni-erlangen.de

    2014-09-15

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.

  11. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    International Nuclear Information System (INIS)

    Mann, Melanie C.; Strobel, Sarah; Fleckenstein, Bernhard; Kress, Andrea K.

    2014-01-01

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo

  12. The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway

    International Nuclear Information System (INIS)

    Angeline, Michael; Merle, Eric; Moroianu, Junona

    2003-01-01

    E7, the major transforming protein of high-risk human papillomavirus (HPV), type 16, binds and inactivates the retinoblastoma protein (pRb), and the Rb-related proteins p107 and p130. HPV16 E7 is a nuclear protein lacking a classical basic nuclear localization signal. In this study we investigated the nuclear import of HPV16 E7 oncoprotein in digitonin-permeabilized HeLa cells. HPV16 E7 nuclear import was independent of pRb, as an E7 ΔDLYC variant defective in pRb binding was imported into the nuclei of digitonin-permeabilized cells as efficiently as wild-type E7 in the presence of exogenous cytosol. Interestingly, we discovered that HPV16 E7 is imported into the nuclei via a novel pathway different from those mediated by Kap α2β1 heterodimers, Kap β1, or Kap β2. Nuclear accumulation of E7 required Ran and was not inhibited by the RanG19V-GTP variant, an inhibitor of Kap β mediated import pathways. Together the data suggest that HPV16 E7 translocates through the nuclear pores via a nonclassical Ran-dependent pathway, independent of the main cytosolic Kap β import receptors

  13. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    International Nuclear Information System (INIS)

    Zacapala-Gómez, Ana Elvira; Del Moral-Hernández, Oscar; Villegas-Sepúlveda, Nicolás; Hidalgo-Miranda, Alfredo; Romero-Córdoba, Sandra Lorena

    2016-01-01

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  14. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    Energy Technology Data Exchange (ETDEWEB)

    Zacapala-Gómez, Ana Elvira, E-mail: zak_ana@yahoo.com.mx [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Del Moral-Hernández, Oscar, E-mail: odelmoralh@gmail.com [Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Gro., México (Mexico); Villegas-Sepúlveda, Nicolás, E-mail: nvillega@cinvestav.mx [Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México, D.F., México (Mexico); Hidalgo-Miranda, Alfredo, E-mail: ahidalgo@inmegen.gob.mx [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); Romero-Córdoba, Sandra Lorena, E-mail: sromero_cordoba@hotmail.com [Laboratorio de Genómica del Cáncer, Instituto Nacional de Medicina Genómica (INMEGEN), México, D.F., México (Mexico); and others

    2016-01-15

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  15. Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins

    Energy Technology Data Exchange (ETDEWEB)

    Onder, Zeynep; Moroianu, Junona, E-mail: moroianu@bc.edu

    2014-01-20

    We have previously discovered and characterized the nuclear import pathways for the E7 oncoproteins of mucosal alpha genus HPVs, type 16 and 11. Here we investigated the nuclear import of cutaneous beta genus HPV8 E7 protein using confocal microscopy after transfections of HeLa cells with EGFP-8E7 and mutant plasmids and nuclear import assays in digitonin-permeabilized HeLa cells. We determined that HPV8 E7 contains a nuclear localization signal (NLS) within its zinc-binding domain that mediates its nuclear import. Furthermore, we discovered that a mostly hydrophobic patch {sub 65}LRLFV{sub 69} within the zinc-binding domain is essential for the nuclear import and localization of HPV8 E7 via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153. Substitution of the hydrophobic residues within the {sub 65}LRLFV{sub 69} patch to alanines, and not R66A mutation, disrupt the interactions between the 8E7 zinc-binding domain and Nup62 and Nup153 and consequently inhibit nuclear import of HPV8 E7. - Highlights: • HPV8 E7 has a cNLS within its zinc-binding domain that mediates its nuclear import. • Discovery of a hydrophobic patch that is critical for the nuclear import of HPV8 E7. • HPV8 E7 nuclear import is mediated by hydrophobic interactions with FG-Nups, Nup62 and Nup153.

  16. Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

    Science.gov (United States)

    Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

    2014-01-01

    Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

  17. Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

    Science.gov (United States)

    Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

  18. PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-Ibeta/SET.

    Science.gov (United States)

    Anazawa, Yoshio; Nakagawa, Hidewaki; Furihara, Mutsuo; Ashida, Shingo; Tamura, Kenji; Yoshioka, Hiroki; Shuin, Taro; Fujioka, Tomoaki; Katagiri, Toyomasa; Nakamura, Yusuke

    2005-06-01

    Through genome-wide cDNA microarray analysis coupled with microdissection of prostate cancer cells, we identified a novel gene, prostate collagen triple helix (PCOTH), showing overexpression in prostate cancer cells and its precursor cells, prostatic intraepithelial neoplasia (PIN). Immunohistochemical analysis using polyclonal anti-PCOTH antibody confirmed elevated expression of PCOTH, a 100-amino-acid protein containing collagen triple-helix repeats, in prostate cancer cells and PINs. Knocking down PCOTH expression by small interfering RNA (siRNA) resulted in drastic attenuation of prostate cancer cell growth, and concordantly, LNCaP derivative cells that were designed to constitutively express exogenous PCOTH showed higher growth rate than LNCaP cells transfected with mock vector, suggesting the growth-promoting effect of PCOTH on prostate cancer cell. To investigate the biological mechanisms of this growth-promoting effect, we applied two-dimensional differential gel electrophoresis (2D-DIGE) to analyze the phospho-protein fractions in LNCaP cells transfected with PCOTH. We found that the phosphorylation level of oncoprotein TAF-Ibeta/SET was significantly elevated in LNCaP cells transfected with PCOTH than control LNCaP cells, and these findings were confirmed by Western blotting and in-gel kinase assay. Furthermore, knockdown of endogenous TAF-Ibeta expression by siRNA also attenuated viability of prostate cancer cells as well. These findings suggest that PCOTH is involved in growth and survival of prostate cancer cells thorough, in parts, the TAF-Ibeta pathway, and that this molecule should be a promising target for development of new therapeutic strategies for prostate cancers.

  19. Evaluation of tumor markers (HER-2/neu oncoprotein, CEA, and CA 15.3) in patients with locoregional breast cancer: prognostic value.

    Science.gov (United States)

    Molina, Rafael; Augé, Jose M; Escudero, Jose M; Filella, Xavier; Zanon, Gabriel; Pahisa, Jaume; Farrus, Blanca; Muñoz, Montserrat; Velasco, Martin

    2010-06-01

    Tumor markers were studied in the sera of 883 untreated patients with primary breast cancer diagnosed between 1989 and 2007. Abnormal human epidermal growth factor receptor 2 (HER-2)/neu levels (>15 ng/mL) were found in 9.5%, carcinoembryonic antigen (CEA) in 15.9%, and cancer antigen (CA) 15.3 in 19.7% of the patients. One or more tumor markers were abnormal in 305 (34.5%) of the 883 studied patients. Significantly higher serum HER-2/neu levels were found in patients with tissue overexpression of this oncoprotein (p CEA, CA 15.3, and HER-2/neu (only in those patients with tissue overexpression) serum levels were related with tumor stage (tumor size and nodal involvement) and steroid receptors (higher values in estrogen receptor-negative (ER-) tumors). Univariate analysis showed that HER-2/neu serum levels were prognostic factors in disease-free survival (DFS) and overall survival (OS) only in patients with tissue overexpression. Multivariate analysis in 834 patients show that nodal involvement, tumor size, ER, CEA, and adjuvant treatment were independent prognostic factors in DFS and OS. When only patients with HER-2/neu overexpression in tissue were studied, tumor size, nodal involvement, and tumor markers (one or another positive) were independent prognostic factors for both DFS and OS. HER-2/neu serum levels were also an independent prognostic factor, with CEA, ER, and nodes in 106 patients treated with neoadjuvant treatment. In summary, serum HER-2/neu, CEA, and CA 15.3 are useful tools in the prognostic evaluation of patients with primary breast cancer.

  20. Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein

    Science.gov (United States)

    Marsh, Elizabeth K.; Delury, Craig P.; Davies, Nicholas J.; Weston, Christopher J.; Miah, Mohammed A.L.; Banks, Lawrence; Parish, Joanna L.

    2017-01-01

    The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification. PMID:28061478

  1. ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

    Science.gov (United States)

    Lopez, M; Oettgen, P; Akbarali, Y; Dendorfer, U; Libermann, T A

    1994-05-01

    The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

  2. CAPERalpha is a novel Rel-TAD-interacting factor that inhibits lymphocyte transformation by the potent Rel/NF-kappaB oncoprotein v-Rel.

    Science.gov (United States)

    Dutta, Jui; Fan, Gaofeng; Gélinas, Céline

    2008-11-01

    The Rel/NF-kappaB transcription factors are constitutively activated in many human cancers. The Rel proteins in this family are implicated in leukemia/lymphomagenesis, but the mechanism is not completely understood. Previous studies showed that the transcription activation domains (TADs) of the viral oncoprotein v-Rel and its cellular Rel/NF-kappaB homologues c-Rel and RelA are key determinants of their different transforming activities in primary lymphocytes. Substitution of a Rel TAD for that of RelA conferred a strong transforming phenotype upon RelA, which otherwise failed to transform cells. To gain insights into protein interactions that influence cell transformation by the Rel TADs, we identified factors that interact with the TAD of v-Rel, the most oncogenic member of the Rel/NF-kappaB family. We report that the coactivator for transcription factors AP-1 and estrogen receptors, CAPERalpha, interacts with the v-Rel TAD and potently synergizes v-Rel-mediated transactivation. Importantly, coexpression of CAPERalpha markedly reduced and delayed v-Rel's transforming activity in primary lymphocytes, whereas a dominant-negative mutant enhanced the kinetics of v-Rel-mediated transformation. Furthermore, small interfering RNA-mediated knockdown of CAPERalpha in v-Rel-transformed lymphocytes significantly enhanced colony formation in soft agar. Since the potency of Rel-mediated transactivation is an important determinant of lymphocyte transformation, as is Rel's ability to induce transcriptional repression, these data suggest that CAPERalpha's interaction with the Rel TAD could modulate Rel/NF-kappaB's transforming activity by facilitating expression or dampening repression of specific gene subsets important for oncogenesis. Overall, this study identifies CAPERalpha as a new transcriptional coregulator for v-Rel and reveals an important role in modulating Rel's oncogenic activity.

  3. Discovery of multiple interacting partners of gankyrin, a proteasomal chaperone and an oncoprotein--evidence for a common hot spot site at the interface and its functional relevance.

    Science.gov (United States)

    Nanaware, Padma P; Ramteke, Manoj P; Somavarapu, Arun K; Venkatraman, Prasanna

    2014-07-01

    Gankyrin, a non-ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin-proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line- MDA-MB-435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA-MB-231 cells in which the endogenous CLIC1 is silenced, trans-expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein-protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. © 2013 Wiley Periodicals, Inc.

  4. Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

    Science.gov (United States)

    Marsh, Elizabeth K; Delury, Craig P; Davies, Nicholas J; Weston, Christopher J; Miah, Mohammed A L; Banks, Lawrence; Parish, Joanna L; Higgs, Martin R; Roberts, Sally

    2017-03-21

    The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

  5. Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

    Science.gov (United States)

    Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

    2016-12-27

    The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2 S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2 Y393F ) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2 Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2 Y393F/S394A mice and Mdm2 S394A mice display similar phenotypes.

  6. Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

    Science.gov (United States)

    Allen, Cory; McDonald, Cari; Giannini, Caterina; Peng, Kah Whye; Rosales, Gabriela; Russell, Stephen J; Galanis, Evanthia

    2004-11-01

    Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP

  7. Differential expression of galectin-3, CK19, HBME1, and Ret oncoprotein in the diagnosis of thyroid neoplasms by fine needle aspiration biopsy

    Directory of Open Access Journals (Sweden)

    Saleh Husain

    2009-01-01

    Full Text Available Background: Fine needle aspiration biopsy (FNAB is a common and excellent procedure for the evaluation of thyroid lesions that require surgical resection. At times, the FNAB diagnosis can be difficult, particularly of follicular-patterned lesions. Previous studies have shown that some immunohistochemical (IHC markers may be helpful in establishing more accurate diagnosis. In this study, our goal was to evaluate four of the recently investigated markers in differentiating benign from malignant thyroid nodules on FNABs. Materials and Methods: We performed IHC staining of galectin-3, Ret oncoprotein (Ret, HBME-1, and cytokeratin 19 (CK19, on cell block sections of thyroid FNAB cases that had corresponding surgical resections. They included 44 benign lesions (37 hyperplastic or cellular nodules, HN; and 7 follicular adenomas, FA and 27 malignant tumors (6 follicular carcinoma, FC; 19 classic papillary carcinoma, PTC; and 2 follicular variant of papillary carcinoma, FVPC. The stains were done according to the standard avidin-biotin-peroxidase method. Results: Statistical analysis showed that immunoexpression was significantly higher in the malignant group for all four markers. The sensitivity for positive expression for all benign lesions versus malignant tumors was as follows: 10/44 (22.7% versus 25/27 (92.6% for galectin-3; 14/44 (31.8% versus 23/27 (85% for Ret; 12/44 (27.3% versus 24/27 (88.8% for HBME-1; and 13/44 (29.5% versus 23/27 (85% for CK19. The sensitivity and specificity was highest for galectin-3 (92.6% and 77.3%, respectively followed by HMBE-1 (88.9% and 72.7%, respectively. When combining the markers′ expressions, the panel of galectin-3 + HBME-1 showed the highest sensitivity and specificity (90.7% and 75%, respectively, but this was, however, lower than galectin-3 alone (92.3% and 77.3%, respectively. Conclusion: We conclude that galectin-3 is the best single marker in differentiating benign from malignant thyroid lesions with

  8. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  9. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

    Energy Technology Data Exchange (ETDEWEB)

    Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States); Ratner, Lee [Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Lairmore, Michael D. [University of California-Davis, School of Veterinary Medicine, One Shields Avenue, Davis, CA 95618 (United States); Martinez, Ernest [Department of Biochemistry, University of California, Riverside, CA 92521 (United States); Lüscher, Bernhard [Institute of Biochemistry, Klinikum, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen (Germany); Robson, Craig N. [Northern Institute for Cancer Research, Newcastle University, The Medical School, Newcastle upon Tyne, NE2 4HH (United Kingdom); Henriksson, Marie [Department of Microbiology, Cell and Tumor Biology, Karolinska Institutet, Stockholm (Sweden); Harrod, Robert, E-mail: rharrod@smu.edu [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States)

    2015-02-15

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30{sup II} interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

  10. Prevalence of human papilloma virus with risk of cervical cancer among south Indian women: A genotypic study with meta-analysis and molecular dynamics of HPV E6 oncoprotein.

    Science.gov (United States)

    Akram Husain, R S; Rajakeerthana, R; Sreevalsan, Anoop; Prema Jayaprasad, P; Ahmed, Shiek S S J; Ramakrishnan, V

    2018-04-23

    Cervical cancer (CC) is a major fatal health problem in women with high mortality worldwide. Human Papilloma Virus (HPV) is considered as one of the causative factors for CC. The HPV prevalence and their genotype distribution among women population are essential to evaluate the deteriorating impact of HPV. A cross-sectional study was performed involving 212 participants to identify the prevalence of high-risk HPV genotypes in south India using PCR and DNA Sequencing. The results obtained from cross-sectional study were used to conduct a meta-analysis of the previous published studies on HPV prevalence and genotype distribution across six geographical regions (North, Northeast, East, Central, West, and South) of India. Additionally, molecular simulation was performed using GROMACS software to determine the structural differences of E6 oncoprotein in HPV-16 and 18 genotypes, characterized from Indian subjects. Among the study participants, the HPV prevalence was found to be 81.70% in CC, 71.42% in HSIL and 61.30% in LSIL. The meta-analysis showed a high prevalence of HPV-16 in CC across the entire six regions. Of which, South and North India were found to have high HPV prevalence among Indian regions. Further, simulation of E6 oncoprotein revealed structural differences between HPV-16 and 18 which may be associated with their oncogenic nature. The HPV-16 and 18 were noticed to be highly prevalent in Indian women. Health awareness and vaccination programs are regularly needed to protect Indian women community. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

    Science.gov (United States)

    Krishnapuram, Rashmi; Dhurandhar, Emily J; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V

    2013-01-01

    Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

  12. Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

    Directory of Open Access Journals (Sweden)

    Rashmi Krishnapuram

    Full Text Available Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1. Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

  13. Combinatorial therapy with adenoviral-mediated PTEN and a PI3K inhibitor suppresses malignant glioma cell growth in vitro and in vivo by regulating the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Nan, Yang; Guo, Liyun; Song, Yunpeng; Wang, Le; Yu, Kai; Huang, Qiang; Zhong, Yue

    2017-08-01

    Glioblastoma is a highly invasive and challenging tumor of the central nervous system. The mutation/deletion of the tumor suppressor phosphatase and tensin homolog (PTEN) gene is the main genetic change identified in glioblastomas. PTEN plays a critical role in tumorigenesis and has been shown to be an important therapeutic target. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is commonly used to inhibit glioma cell growth via regulation of the PI3K/AKT signaling pathway. In this study, we examined the growth inhibitory effects of a combinatorial therapy of adenoviral-mediated PTEN (Ad-PTEN) and LY294002 on LN229 and U251 glioma cells in vitro and on tumor xenografts in vivo. In vitro, LN229 and U251 glioma cells were treated by combinatorial therapy with Ad-PTEN and LY294002. The growth ability was determined by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Cell invasive ability was analyzed by transwell invasion assay and cell apoptosis analysis via FITC-Annexin V analysis. In vivo, U251 subcutaneous glioblastoma xenograft was used to assay anti-tumor effect of combinatorial therapy with Ad-PTEN and LY294002 by mean volume of tumors, immunohistochemistry and TUNEL method. The combinatorial treatment clearly suppressed cell proliferation, arrested the cell cycle, reduced cell invasion and promoted cell apoptosis compared with the Ad-PTEN or LY294002 treatment alone. The treatment worked by inhibiting the PI3K/AKT pathway. In addition, the growth of U251 glioma xenografts treated with the combination of Ad-PTEN and LY294002 was significantly inhibited compared with those treated with Ad-PTEN or LY294002 alone. Our data indicated that the combination of Ad-PTEN and LY294002 effectively suppressed the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach for glioma gene therapy that warrants further investigation.

  14. Detection of HPV and the role of p16INK4A overexpression as a surrogate marker for the presence of functional HPV oncoprotein E7 in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Lardon Filip

    2010-03-01

    Full Text Available Abstract Background Based on the well-recognized etiological role of human papillomavirus (HPV in cervical, anogenital and oropharyngeal carcinogenesis, a potential role of HPV in colorectal carcinogenesis has been suggested. For that reason, the aim of the present study was to investigate the presence of HPV DNA in colorectal carcinomas (CRC and to study overexpression of p16INK4A as a marker for the presence of an active HPV oncoprotein E7. These findings were correlated with clinical and pathological prognostic factors of CRC. Methods The presence of HPV was assessed using a multiplex PCR system of 10 non-biotinylated primers. The amplified fragments of HPV positive samples were further analyzed by a highly sensitive, broad spectrum SPF10 PCR and subsequently genotyped using reverse hybridization in a line probe assay. P16INK4A protein expression was investigated in a subset of 90 (30 HPV positive and 60 HPV negative CRC samples by immunohistochemistry. Results HPV DNA was found in 14.2% of the CRC samples with HPV16 as the most prevalent type. No significant differences in clinical and pathological variables were found between HPV positive and negative CRCs, except for age. HPV positive patients were significantly younger (p = 0.05. There was no significant correlation between the presence of HPV and overexpression of p16INK4A (p = 0.325. Conclusions In conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples may suggest that HPV may be involved in the carcinogenesis of some CRC. However, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16INK4A does not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the role of HPV in CRC.

  15. Stimulation of interleukin-13 expression by human T-cell leukemia virus type 1 oncoprotein Tax via a dually active promoter element responsive to NF-kappaB and NFAT.

    Science.gov (United States)

    Silbermann, Katrin; Schneider, Grit; Grassmann, Ralph

    2008-11-01

    The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein transforms human lymphocytes and is critical for the pathogenesis of HTLV-1-induced adult T-cell leukaemia. In HTLV-transformed cells, Tax upregulates interleukin (IL)-13, a cytokine with proliferative and anti-apoptotic functions that is linked to leukaemogenesis. Tax-stimulated IL-13 is thought to result in autocrine stimulation of HTLV-infected cells and thus may be relevant to their growth. The causal transactivation of the IL-13 promoter by Tax is predominantly dependent on a nuclear factor of activated T cells (NFAT)-binding P element. Here, it was shown that the isolated IL-13 Tax-responsive element (IL13TaxRE) was sufficient to mediate IL-13 transactivation by Tax and NFAT1. However, cyclosporin A, a specific NFAT inhibitor, revealed that Tax transactivation of IL13TaxRE or wild-type IL-13 promoter was independent of NFAT and that NFAT did not contribute to IL-13 upregulation in HTLV-transformed cells. By contrast, Tax stimulation was repressible by an efficient nuclear factor (NF)-kappaB inhibitor (IkBaDN), indicating the requirement for NF-kappaB. The capacity of NF-kappaB to stimulate IL13TaxRE was demonstrated by a strong response to NF-kappaB in reporter assays and by direct binding of NF-kappaB to IL13TaxRE. Thus, IL13TaxRE in the IL-13 promoter represents a dually active promoter element responsive to NF-kappaB and NFAT. Together, these results indicate that Tax causes IL-13 upregulation in HTLV-1-infected cells via NF-kappaB.

  16. Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

    Science.gov (United States)

    Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

    2004-07-01

    Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

  17. Clinical adenoviral gene therapy for prostate cancer

    Czech Academy of Sciences Publication Activity Database

    Schenk, E.; Essand, M.; Bangma, Ch. H.; Barber, Ch.; Behr, J.-P.; Briggs, S.; Carlisle, R.; Cheng, W.-S.; Danielsson, A.; Dautzenberg, I. J. C.; Dzojic, H.; Erbacher, P.; Fisher, K.; Frazier, A.; Georgopoulos, L. J.; Hoeben, R.; Kochanek, S.; Koppers-Lalic, D.; Kraaij, R.; Kreppel, F.; Lindholm, L.; Magnusson, M.; Maitland, N.; Neuberg, P.; Nilsson, B.; Ogris, M.; Remy, J.-S.; Scaife, M.; Schooten, E.; Seymour, L.; Totterman, T.; Uil, T. G.; Ulbrich, Karel; Veldhoven-Zweistra, J. L. M.; de Vrij, J.; van Weerden, W.; Wagner, E.; Willemsen, R.

    2010-01-01

    Roč. 21, č. 7 (2010), s. 807-813 ISSN 1043-0342 EU Projects: European Commission(XE) 512087 - GIANT Keywords : adenovirus * gene delivery * prostate cancer Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.829, year: 2010

  18. Use of adenoviral vectors as veterinary vaccines.

    Science.gov (United States)

    Ferreira, T B; Alves, P M; Aunins, J G; Carrondo, M J T

    2005-10-01

    Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

  19. The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

    International Nuclear Information System (INIS)

    Pearson, Joel D; Mohammed, Zubair; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2012-01-01

    ability of this oncoprotein to signal. This is the first study demonstrating that the expression of immunophilin family co-chaperones is promoted by an oncogenic tyrosine kinase. Moreover, this is the first report establishing an important role for Cyp40 in lymphoma

  20. Acute dose and low dose-rate irradiation of carcinoma cells expressing human papillomavirus E6 and E7 oncoproteins - the significance of p53, Rb and G1 arrest status

    International Nuclear Information System (INIS)

    DeWeese, Theodore L.; Walsh, Jonathan C.; Dillehay, Larry E.; Shao, Y.; Kessis, Theodore D.; Cho, Kathleen R.; Nelson, William G.

    1995-01-01

    Purpose: The development of carcinomas in a number of sites including the cervix, vulva and anus have been associated with cellular infection by human papillomaviruses (HPV), including HPV 16 and HPV 18. The mechanism by which these viruses contribute to tumor development or progression seems in part to be related to the integration of the viral genome into the host cells DNA, and the binding of p53 protein by the HPV E6 oncoprotein as well as the binding of the retinoblastoma (Rb) protein and Rb-like proteins by the HPV E7 oncoprotein. These interactions lead to loss of p53 and Rb function including loss of the G 1 cell cycle checkpoint. Although it is believed that both p53 and Rb play a role in the radiosensitivity of the cell, whether alteration in either protein enhances or diminishes cellular radiation response is not clear from the literature. Because HPV-associated tumors such as cervical cancer are often treated with acute dose and/or low dose-rate radiation, we set out to evaluate the radiation response of several carcinoma cell sublines expressing either oncogenic E6 or E7 to both types of radiation, and to determine if p53/Rb dependent G 1 arrest is an important determinant of cell fate after irradiation. Materials and Methods: We have previously developed a series of RKO colorectal carcinoma cell sublines expressing both low-risk (HPV 11) and high-risk (HPV 16) E6 and E7 genes. p53-dependent G 1 arrest is intact in RKO parental cells and cells expressing low-risk E6 proteins, while the G 1 arrest is abrogated in cells expressing high-risk E6 or E7. Clonogenic survival was assessed after exposure to acute dose (1 Gy/min) and low dose-rate (0.25 Gy/hour) radiation. The radiobiologic parameters α, β and the surviving fraction at 2 Gy (SF2) were determined. SDS-PAGE/immunoblotting was carried out to assess both p53 and p21 WAF1/CIP1 levels after exposure to radiation. Flow cytometry was performed before and after exposure to low dose-rate radiation to

  1. Evaluating the role of CRM1-mediated export for adenovirus gene expression

    International Nuclear Information System (INIS)

    Carter, Christoph C.; Izadpanah, Reza; Bridge, Eileen

    2003-01-01

    A complex of the Adenovirus (Ad) early region 1b 55-kDa (E1b-55kDa) and early region 4 ORF6 34-kDa (E4-34kDa) proteins promotes viral late gene expression. E1b-55kDa and E4-34kDa have leucine-rich nuclear export signals (NESs) similar to that of HIV Rev. It was proposed that E1b-55kDa and/or E4-34kDa might promote the export of Ad late mRNA via their Rev-like NESs, and the transport receptor CRM1. We treated infected cells with the cytotoxin leptomycin B to inhibit CRM1-mediated export; treatment initially delays the onset of late gene expression, but this activity completely recovers as the late phase progresses. We find that the E1b-55kDa NES is not required to promote late gene expression. Previous results showed that E4-34kDa-mediated late gene expression does not require an intact NES (J. Virol. 74 (2000), 6684-6688). Our results indicate that these Ad regulatory proteins promote late gene expression without intact NESs or active CRM1

  2. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    2015-11-01

    Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

  3. Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts.

    NARCIS (Netherlands)

    Geoerger, B; Grill, J; Opolon, P; Morizet, J; Aubert, G; Lecluse, Y; Beusechem-Kaptein, van V.W.; Gerritsen, W.R.; Kirn, DH; Vassal, G

    2003-01-01

    In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015

  4. Detecção imunoistoquímica das oncoproteínas p21ras, c-myc E p53 no carcinoma hepatocelular e no tecido hepático não-neoplásico Immunohistochemical detection of p21ras, c-myc and p53 oncoproteins in hepatocellular carcinoma and in non-neoplastic liver tissue

    Directory of Open Access Journals (Sweden)

    Vera Lucia Nunes Pannain

    2004-12-01

    oncoproteins in hepatocellular carcinoma and non neoplastic tissue. Association of the immunoreactivity of these markers with histological grades and patterns, hepatitis B and C were additionally studied. METHODS: Detection of oncoproteins p21ras, c-myc and p53 was performed immunohistochemically in hepatocellular carcinoma (47 cases and surrounding non neoplastic liver tissue (40 cases. RESULTS: Oncoproteins p21ras, c-myc and p53 were detected in 44,7%, 53,2% and 36,2% of the hepatocellular carcinoma cases, respectively. The p21ras and c-myc immunoreactivity has shown a significant association. However there was no association of p21ras, c-myc and p53 detection with hepatitis B and C virus infections, histological grades and patterns. The same significant association between p21ras and c-myc was observed in non-neoplastic tissue with cirrhosis when compared with tissue without it. The p53 immunoreactivity was negative in all non-neoplastic liver tissue samples. CONCLUSIONS: The immunoreactivity detection of p21ras, c-myc and p53 corroborates previous evidence of their detection in hepatocellular carcinoma that suggest the participation of these proteins in human hepatocarcinogenesis. The significant association between p21ras and c-myc oncoproteins in hepatocellular carcinoma and in cirrhosis can point to an interaction between them mainly, in hepatocarcinogenesis that occurs through cirrhosis.

  5. Combined distemper-adenoviral pneumonia in a dog

    Science.gov (United States)

    Rodriguez-Tovar, Luis E.; Ramírez-Romero, Rafael; Valdez-Nava, Yazel; Nevárez-Garza, Alicia M.; Zárate-Ramos, Juan J.; López, Alfonso

    2007-01-01

    A 3 1/2-month-old pug with oculonasal discharge and seizures was submitted for postmortem examination. Grossly, the lungs had cranioventral consolidation, and microscopically, 2 distinct types of inclusion bodies compatible with Canine distemper virus and Canine adenovirus type 2. Presence of both viruses was confirmed via immunohistochemical staining. PMID:17616064

  6. Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

    Science.gov (United States)

    Osada, Takuya; Morse, Michael A; Hobeika, Amy; Lyerly, H Kim

    2012-06-01

    Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Coinfections of Multiple Virulent Adenoviral Species in Previously Vaccinated Patients

    National Research Council Canada - National Science Library

    Vora, Gary J; Lin, Baochuan; Gratwick, Kevin; Meador, Carolyn; Seto, Donald; Purkayastha, Anjan; Freed, Nikki E; Russell, Kevin; Metzgar, David

    2004-01-01

    .... Despite the general success of the vaccination program, surveillance still detected breakthrough infections cases of acute respiratory disease associated with the adenovirus serotypes (4 and 7...

  8. Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients.

    Science.gov (United States)

    Morse, Michael A; Chaudhry, Arvind; Gabitzsch, Elizabeth S; Hobeika, Amy C; Osada, Takuya; Clay, Timothy M; Amalfitano, Andrea; Burnett, Bruce K; Devi, Gayathri R; Hsu, David S; Xu, Younong; Balcaitis, Stephanie; Dua, Rajesh; Nguyen, Susan; Balint, Joseph P; Jones, Frank R; Lyerly, H Kim

    2013-08-01

    First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.

  9. Nanoscale Proteomic Analysis of Oncoproteins in Hematopoietic Cancers

    Science.gov (United States)

    2012-05-01

    can be measured. Quantitation of AKT2 phosphorylated and unphosphorylated protein peaks was performed using Compass (version 1.8.0) analysis soft...5a SDf → PD 12 112 81F RAEB-2 Highh 5d SDg → PD 14+ 117 75F RAEB-2 Highh 3c,e PD skin (mCR) Yes 5 121 80M RAEB-T Highh 1c PD Yes 3 122 76F RAEB-1

  10. MYCN: from oncoprotein to tumor-associated antigen

    International Nuclear Information System (INIS)

    Pistoia, Vito; Morandi, Fabio; Pezzolo, Annalisa; Raffaghello, Lizzia; Prigione, Ignazia

    2012-01-01

    MYCN is a well-known oncogene over-expressed in different human malignancies including neuroblastoma (NB), rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor, and small cell lung cancer. In the case of NB, MYCN amplification is an established biomarker of poor-prognosis. MYCN belongs to a family of transcription factors (the most important of which is C-MYC) that show a high degree of homology. Down-regulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets. Pre-requisites for a candidate tumor-associated antigen (TAA) to be targeted by immunotherapeutic approaches are the following, (i) expression should be tumor-restricted, (ii) the putative TAA should be up-regulated in cancer cells, and (iii) protein should be processed into immunogenic peptides capable of associating to major histocompatibility complex molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and up-regulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or HLA-A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB. Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTLs) and will be here discussed are the following, (i) the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA class I molecules, the lack of co-stimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and (ii) the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g., soluble MICA and HLA-G in the case of NB) or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the strategy used to generate CTL.

  11. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

    Directory of Open Access Journals (Sweden)

    Barry M Zee

    Full Text Available Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC, which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.

  12. human papillomaviruses E6 and E7 oncoproteins

    Indian Academy of Sciences (India)

    Unknown

    Recent work has uncovered new cellular partners for these .... Hence it is evident that a fine balance exists between these that govern ... still suggest that E7 can have a more global effect on the general .... towards malignancy), increases life span of the cells. E6 ..... nism when expressed alone in HPV positive/negative cell.

  13. Evaluation of Molecular Inhibitors of the c-Myc Oncoprotein

    National Research Council Canada - National Science Library

    Prochownik, Edward V

    2005-01-01

    .... All of these functions require that C-Myc physically associate with another protein. Max. Example of diseases in which c-Myc deregulation occurs include breast cancer (approx. 30% of cases), colon cancer (>85...

  14. MYCN: From Oncoprotein To Tumor-Associated Antigen

    Directory of Open Access Journals (Sweden)

    Vito ePistoia

    2012-11-01

    Full Text Available MYCN is a well known oncogene overexpressed in different human malignancies including neuroblastoma, rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor and small cell lung cancer. In the case of neuroblastoma (NB, MYCN amplification is an established biomarker of poor prognosis. MYCN belongs to a family of transcription factors (the most important of which is CMYC that show a high degree of homology. Downregulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets.Pre-requisites for a candidate tumor-associated antigen (TAA to be targeted by immunotherapeutic approaches are the following, i expression should be tumor-restricted, ii the putative TAA should be up-regulated in cancer cells and iii protein should be processed into immunogenic peptides capable of associating to MHC molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and upregulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or –A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB.Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTL and will be here discussed are the following, i the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA- class I molecules, the lack of costimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and ii the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g. soluble MICA and HLA-G in the case of NB or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the strategy used to generate CTL

  15. MYCN: from oncoprotein to tumor-associated antigen

    Energy Technology Data Exchange (ETDEWEB)

    Pistoia, Vito; Morandi, Fabio; Pezzolo, Annalisa; Raffaghello, Lizzia; Prigione, Ignazia, E-mail: vitopistoia@ospedale-gaslini.ge.it [Laboratory of Oncology, Translational Research and Laboratory Medicine, G. Gaslini Institute, Genoa (Italy)

    2012-11-16

    MYCN is a well-known oncogene over-expressed in different human malignancies including neuroblastoma (NB), rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor, and small cell lung cancer. In the case of NB, MYCN amplification is an established biomarker of poor-prognosis. MYCN belongs to a family of transcription factors (the most important of which is C-MYC) that show a high degree of homology. Down-regulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets. Pre-requisites for a candidate tumor-associated antigen (TAA) to be targeted by immunotherapeutic approaches are the following, (i) expression should be tumor-restricted, (ii) the putative TAA should be up-regulated in cancer cells, and (iii) protein should be processed into immunogenic peptides capable of associating to major histocompatibility complex molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and up-regulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or HLA-A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB. Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTLs) and will be here discussed are the following, (i) the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA class I molecules, the lack of co-stimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and (ii) the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g., soluble MICA and HLA-G in the case of NB) or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the strategy used to generate CTL.

  16. ETS-1 oncoprotein expression is decreased in aggressive papillary ...

    African Journals Online (AJOL)

    E.A. Ibrahim

    2016-10-19

    Oct 19, 2016 ... aggressive papillary transitional cell carcinoma of the urinary bladder: An immunohistochemical study. E.A. Ibrahim. ∗. , M.R. Hassan, S.A. Sammour. Pathology Department, Faculty of Medicine, Ain Shams University, Egypt. Received 23 August 2015; received in revised form 11 October 2015; accepted 12 ...

  17. Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

    DEFF Research Database (Denmark)

    Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy

    2013-01-01

    to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance...... is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription...

  18. Cervical Cancers Require the Continuous Expression of the Human Papillomavirus Type 16 E7 Oncoprotein Even in the Presence of the Viral E6 Oncoprotein

    Science.gov (United States)

    Jabbar, Sean F.; Park, Soyeong; Schweizer, Johannes; Berard-Bergery, Marthe; Pitot, Henry C.; Lee, Denis; Lambert, Paul F.

    2012-01-01

    High-risk human papillomaviruses (HPV), such as HPV-16, are etiologic agents of a variety of anogenital and oral malignancies, including nearly all cases of cervical cancer. Cervical cancers arising in transgenic mice that express HPV-16 E7 in an inducible manner require the continuous expression of E7 for their maintenance. However, in HPV-associated cancers in vivo, E6 and E7 invariably are co-expressed. In this study, we investigated whether cervical cancers rely on the continuous expression of E7 in the context of constitutively expressed E6. We placed the inducible HPV-16 E7 transgene onto a background in which HPV-16 E6 was constitutively expressed. In transgenic mice with high-grade cervical dysplastic lesions and cervical cancer, repressing the expression of E7 led to the regression of all cancers and the vast majority of high-grade dysplastic lesions. In addition, cervical cancers were occasionally observed in transgenic mice in which E7 was repressed and then re-expressed. Our findings therefore indicate that even in the presence of constitutively expressed E6, the continuous expression of E7 is required for the maintenance of cervical cancers and most precancerous lesions. These data have important implications for the potential clinical use of drugs designed to inhibit the expression and/or function of E7 to treat HPV-associated cancers. PMID:22700879

  19. Characterization of adenoviral transduction profile in prostate cancer cells and normal prostate tissue.

    Science.gov (United States)

    Ai, Jianzhong; Tai, Phillip W L; Lu, Yi; Li, Jia; Ma, Hong; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

    2017-09-01

    Prostate diseases are common in males worldwide with high morbidity. Gene therapy is an attractive therapeutic strategy for prostate diseases, however, it is currently underdeveloped. As well known, adeno virus (Ad) is the most widely used gene therapy vector. The aims of this study are to explore transduction efficiency of Ad in prostate cancer cells and normal prostate tissue, thus further providing guidance for future prostate pathophysiological studies and therapeutic development of prostate diseases. We produced Ad expressing enhanced green fluorescence protein (EGFP), and characterized the transduction efficiency of Ad in both human and mouse prostate cancer cell lines in vitro, as well as prostate tumor xenograft, and wild-type mouse prostate tissue in vivo. Ad transduction efficiency was determined by EGFP fluorescence using microscopy and flow cytometry. Cell type-specific transduction was examined by immunofluorescence staining of cell markers. Our data showed that Ad efficiently transduced human and mouse prostate cancer cells in vitro in a dose dependent manner. Following intratumoral and intraprostate injection, Ad could efficiently transduce prostate tumor xenograft and the major prostatic cell types in vivo, respectively. Our findings suggest that Ad can efficiently transduce prostate tumor cells in vitro as well as xenograft and normal prostate tissue in vivo, and further indicate that Ad could be a potentially powerful toolbox for future gene therapy of prostate diseases. © 2017 Wiley Periodicals, Inc.

  20. Adenoviral gene transfer of angiostatic ATF-BPTI inhibits tumour growth

    International Nuclear Information System (INIS)

    Lefesvre, Pierre; Attema, Joline; Bekkum, Dirk van

    2002-01-01

    The outgrowth of new vessels – angiogenesis – in the tumour mass is considered to be a limiting factor of tumour growth. To inhibit the matrix lysis that is part of the tumour angiogenesis, we employed the chimeric protein mhATF-BPTI, composed of the receptor binding part of the urokinase (ATF) linked to an inhibitor of plasmin (BPTI). For delivery, recombinant adenovirus encoding the transgene of interest was injected intravenously or locally into the tumour. The anti tumour effect of this compound was compared to that of human endostatin and of mhATF alone in two different rat bronchial carcinomas growing either as subcutaneous implants or as metastases. Significant inhibition of the tumour growth and decrease of the number of lung metastasis was achieved when the concentration of mhATF-BPTI at the tumour site was above 400 of ng / g tissue. This concentration could be achieved via production by the liver, only if permissive to the recombinant adenovirus. When the tumour cells could be transduced, local delivery of the vector was enough to obtain a response. In the case of metastasis, the capacity of the lung tissue to concentrate the encoded protein was essential to reach the required therapeutic levels. Further, endostatin or mhATF could not reproduce the effects of mhATF-BPTI, at similar concentrations (mhATF) and even at 10-fold higher concentration (endostatin). The ATF-BPTI was shown to inhibit tumour growth of different rat lung tumours when critical concentration was reached. In these tumour models, endostatin or ATF induce almost no tumour response

  1. Characterization of infectivity of knob-modified adenoviral vectors in glioma

    NARCIS (Netherlands)

    C.P.L. Paul (C. P L); M. Everts (M.); J.N. Glasgow (J.); P. Dent (P.); P.B. Fisher (P.); I.V. Ulasov (I.); M.S. Lesniak (M.); M.A. Stoff-Khalili (M.); J.C. Roth (J.); M. Preuss (Michael); C.M.F. Dirven (Clemens); M.L.M. Lamfers (Martine); T. Siegal (Tali); Z.B. Zhu (Z.); R.E. Curiel (Rafael E.)

    2008-01-01

    textabstractMalignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors, particularly vectors derived

  2. Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector

    OpenAIRE

    Sasaki, Makoto; Mathis, J Michael; Jennings, Merilyn H; Jordan, Paul; Wang, Yuping; Ando, Tomoaki; Joh, Takashi; Alexander, J Steven

    2005-01-01

    Abstract Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammat...

  3. Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

    Directory of Open Access Journals (Sweden)

    Xishuang Liu

    2011-01-01

    Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

  4. Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

    Science.gov (United States)

    Weiss, Yoram G; Maloyan, Alina; Tazelaar, John; Raj, Nichelle; Deutschman, Clifford S

    2002-09-01

    The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

  5. Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors

    NARCIS (Netherlands)

    Verhaagh, S.; Jong, E. de; Goudsmit, J.; Lecollinet, S.; Gillissen, G.; Vries, M. de; Leuven, K. van; Que, I.; Ouwehand, K.; Mintardjo, R.; Weverling, G.J.; Radošević, K.; Richardson, J.; Eloit, M.; Lowik, C.; Quax, P.; Havenga, M.

    2006-01-01

    Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here,

  6. ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

    Directory of Open Access Journals (Sweden)

    Lacher Markus D

    2011-07-01

    Full Text Available Abstract Background We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9:2088-95 and TGF-β (Cancer Res. 2006 Feb 1;66(3:1648-57 signaling negatively regulate coxsackie virus and adenovirus receptor (CAR cell-surface expression and adenovirus uptake. In the case of TGF-β, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT, a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration. Results Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic and MDA-MB-231 (breast human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-β-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-β-induced mesenchymal phenotype of PANC-1 cells, TGF-β did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-β may inhibit CAR expression by regulating factor(s that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal CAR promoter is positively regulated through conserved ETS and CRE elements. Conclusions This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.

  7. Infectivity enhanced adenoviral-mediated mda-7/IL-24 gene therapy for ovarian carcinoma

    NARCIS (Netherlands)

    Leath, CA; Kataram, M; Bhagavatula, P; Gopalkrishnan, RV; Dent, P; Fisher, PB; Pereboev, A; Carey, D; Lebedeva, [No Value; Haisma, HJ; Alvarez, RD; Curiel, DT; Mahasreshti, PJ

    Objective. Melanoma differentiation associated gene-7 [mda-7/interleukin (IL)-24] has been identified as a novel anti-cancer agent, which specifically induces apoptosis in cancer cells but not in normal epithelial, endothelial and fibroblast cells. The objective of this study was to evaluate the

  8. Preparation of a recombinant adenoviral encoding human NIS gene and its specific expression in cardiomyocytes

    International Nuclear Information System (INIS)

    Wang Lihua; Zhang Miao; Guo Rui; Shi Shuo; Li Biao

    2012-01-01

    Objective: To construct a recombinant adenovirus vector containing the human NIS gene with the myosin light chain-2(MLC-2v) gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyocytes. Methods: MLC-2v promoter and NIS were subcloned into an adenovirus shuttle vector, and forwarded by homologous recombination in the bacteria BJ5183 containing AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the HEK293 cells to obtain recombinant adenovirus Ad-MLC-NIS. Ad-cytomegalovirus (CMV)-NIS with cytomegalovirus as the promoter, Ad-MLC without NIS and Ad-NIS without promoter were constructed as the controls. Cardiomyocytes and non-cardiomyocytes were then infected by the adenovirus. The protein expression was tested by Western blot analysis. The function and features of NIS protein were evaluated by dynamic iodide uptake and NaClO 4 iodine uptake inhibition test in vitro. The viability and proliferation of cardiomyocytes after adenovirus transfection and radioiodine incubation were checked by trypan blue staining. Results: Recombinant NIS adenovirus was successfully constructed. Western blot analysis showed that the NIS protein was highly expressed in cardiomyocytes transfected with Ad-MLC-NIS, and all cells transfected with Ad-CMV-NIS. However, in non-cardiomyocytes transfected with Ad-MLC-NIS, little NIS protein was detected. Dynamic iodine uptake tests showed that the peaks of iodide uptake of the three different cell lines (H9C2, A549, U87 cell) transfected with Ad-MLC-NIS were 5844.0, 833.6 and 846.0 counts · min -1 , respectively. The iodide uptake function of H9C2 was inhibited by NaClO 4 . There was almost no change in cell viability and proliferation when the MOI was 100. Conclusions: Ad-MLC-NIS allows myocardial specific expression of an external gene, and the cardiomyocytes with NIS expression are capable of iodine uptake. Further research of NIS as a reporter gene in myocardium is needed. (authors)

  9. p21 promotes oncolytic adenoviral activity in ovarian cancer and is a potential biomarker

    Directory of Open Access Journals (Sweden)

    Lockley Michelle

    2010-07-01

    Full Text Available Abstract The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015. We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1 to 0.03 pfu/cell (TOV21G. Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.

  10. Alterations of plasma lipids in mice via adenoviral-mediated hepatic overexpression of human ABCA1

    NARCIS (Netherlands)

    Wellington, Cheryl L.; Brunham, Liam R.; Zhou, Steven; Singaraja, Roshni R.; Visscher, Henk; Gelfer, Allison; Ross, Colin; James, Erick; Liu, Guoqing; Huber, Mary T.; Yang, Yu-Zhou; Parks, Robin J.; Groen, Albert; Fruchart-Najib, Jamila; Hayden, Michael R.

    2003-01-01

    ATP binding cassette transporter A1 (ABCA1) is a widely expressed lipid transporter essential for the generation of HDL. ABCA1 is particularly abundant in the liver, suggesting that the liver may play a major role in HDL homeostasis. To determine how hepatic ABCA1 affects plasma HDL cholesterol

  11. Biotherapeutics as alternatives to antibiotics: Effects of adenoviral delivered cytokines on innate and adaptive immunity

    Science.gov (United States)

    Acceptable alternatives to the use of antibiotics in food animal practice need to be explored. The use of immunomodulators is a promising area for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease during periods of peak disease incidence. We developed a method...

  12. Targeting oncoprotein stability overcomes drug resistance caused by FLT3 kinase domain mutations.

    Directory of Open Access Journals (Sweden)

    Chuanjiang Yu

    Full Text Available FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML. Internal tandem duplications (ITDs in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.

  13. Homologous recombination control by the anti-apoptotic onco-protein Bcl-2

    International Nuclear Information System (INIS)

    Dumay, A.

    2003-12-01

    This research thesis deals with the different biological mechanisms, notably the repair and apoptosis mechanisms induced by irradiation in cells. After a presentation of the genotoxic stress and DNA repair mechanisms, the author discusses the cellular response to a DNA double-strand break, and the regulation of these response mechanisms (how a cellular response emerges: life or death). The next part deals with the apoptosis (cell death by necrosis or apoptosis), and presents the BCL-2 protein family. Results are then reported on laboratory studies of the effect of this protein family

  14. Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family

    International Nuclear Information System (INIS)

    Couceiro, Jose R.; Martin-Bermudo, Maria D.; Bustelo, Xose R.

    2005-01-01

    Vav proteins are phosphorylation-dependent GDP/GTP exchange factors for Rho/Rac GTPases. Despite intense characterization of mammalian Vav proteins both biochemically and genetically, there is little information regarding the conservation of their biological properties in lower organisms. To approach this issue, we have performed a characterization of the regulatory, catalytic, and functional properties of the single Vav family member of Drosophila melanogaster. These analyses have shown that the intramolecular mechanisms controlling the enzyme activity of mammalian Vav proteins are already present in Drosophila, suggesting that such properties have been set up before the divergence between protostomes and deuterostomes during evolution. We also show that Drosophila and mammalian Vav proteins have similar catalytic specificities. As a consequence, Drosophila Vav can trigger oncogenic transformation, morphological change, and enhanced cell motility in mammalian cells. Gain-of-function studies using transgenic flies support the implication of this protein in cytoskeletal-dependent processes such as embryonic dorsal closure, myoblast fusion, tracheal development, and the migration/guidance of different cell types. These results highlight the important roles of Vav proteins in the signal transduction pathways regulating cytoskeletal dynamics. Moreover, they indicate that the foundations for the regulatory and enzymatic activities of this protein family have been set up very early during evolution

  15. Oncoprotein DEK as a tissue and urinary biomarker for bladder cancer

    International Nuclear Information System (INIS)

    Datta, Antara; Adelson, Martin E; Mogilevkin, Yakov; Mordechai, Eli; Sidi, Abraham A; Trama, Jason P

    2011-01-01

    Bladder cancer is a significant healthcare problem in the United States of America with a high recurrence rate. Early detection of bladder cancer is essential for removing the tumor with preservation of the bladder, avoiding metastasis and hence improving prognosis and long-term survival. The objective of this study was to analyze the presence of DEK protein in voided urine of bladder cancer patients as a urine-based bladder cancer diagnostic test. We examined the expression of DEK protein by western blot in 38 paired transitional cell carcinoma (TCC) bladder tumor tissues and adjacent normal tissue. The presence of DEK protein in voided urine was analyzed by western blot in 42 urine samples collected from patients with active TCC, other malignant urogenital disease and healthy individuals. The DEK protein is expressed in 33 of 38 bladder tumor tissues with no expression in adjacent normal tissue. Based on our sample size, DEK protein is expressed in 100% of tumors of low malignant potential, 92% of tumors of low grade and in 71% of tumors of high grade. Next, we analyzed 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals for DEK protein expression by western blot analysis. We are the first to show that the DEK protein is present in the urine of bladder cancer patients. Approximately 84% of TCC patient urine specimens were positive for urine DEK. Based on our pilot study of 38 bladder tumor tissue and 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals; DEK protein is expressed in bladder tumor tissue and voided urine of bladder cancer patients. The presence of DEK protein in voided urine is potentially a suitable biomarker for bladder cancer and that the screening for the presence of DEK protein in urine can be explored as a noninvasive diagnostic test for bladder cancer

  16. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT–SSX, and more than half of SYT–SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT–SSX function, and demonstrated how our new system will help to overcome this issue.

  17. Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage

    International Nuclear Information System (INIS)

    Park, Jung Wook; Nickel, Kwangok P.; Torres, Alexandra D.; Lee, Denis; Lambert, Paul F.; Kimple, Randall J.

    2014-01-01

    Background and purpose: Patients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV−) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV− HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. Material and methods: Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. Results: HPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation. Conclusions: Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV− HNC

  18. Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

    Directory of Open Access Journals (Sweden)

    Mallory E. Harden

    2017-01-01

    Full Text Available The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs. While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.

  19. Human papillomavirus E6 and E7 oncoproteins as risk factors for ...

    Indian Academy of Sciences (India)

    HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or low-risk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign ...

  20. Human papillomavirus E6 and E7 oncoproteins as risk factors

    Indian Academy of Sciences (India)

    Prakash

    J. Biosci. 34(1), March 2009. 1. Introduction. Human papillomavirus (HPV) is a double-stranded DNA virus that ..... contact and loss of cell polarity (Watson et al 2003; Thomas ..... Arrand JR 1995 Translation of the human papillomavirus type 16.

  1. Evaluation of HER2/neu oncoprotein in serum & tissue samples of women with breast cancer

    Directory of Open Access Journals (Sweden)

    Shailaja Shukla

    2016-01-01

    Interpretation & conclusions: The results suggest that elevated serum HER2 level was associated with a clinicopathological aggressive phenotype of breast carcinoma and was related to tissue HER2 overexpression. Therefore, serum HER2 may be useful for monitoring the course of the disease and response to treatment.

  2. The c-myc oncoprotein forms a specific complex with the product of the retinoblastoma gene

    NARCIS (Netherlands)

    Bernards, R.A.; Rustgi, A.K.; Dyson, N.; Hill, D.

    1991-01-01

    Myc proteins are involved in the regulation of cell proliferation and differentiation. Deregulated expression of myc family genes has been implicated in the genesis of a variety of cancers. Myc proteins share significant sequence homology in the carboxyl terminus with a number of

  3. The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers

    DEFF Research Database (Denmark)

    Cohen-Eliav, Michal; Golan-Gerstl, Regina; Siegfried, Zahava

    2013-01-01

    and lung tumors and found that the gene encoding for the splicing factor SRSF6 is amplified and overexpressed in these cancers. Moreover, overexpression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them...

  4. SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

    Science.gov (United States)

    Almeida, Luciana O; Neto, Marinaldo P C; Sousa, Lucas O; Tannous, Maryna A; Curti, Carlos; Leopoldino, Andreia M

    2017-04-18

    Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

  5. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

  6. Bag3-induced autophagy is associated with degradation of JCV oncoprotein, T-Ag.

    Directory of Open Access Journals (Sweden)

    Ilker Kudret Sariyer

    Full Text Available JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML. In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag, in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.

  7. Bag3-induced autophagy is associated with degradation of JCV oncoprotein, T-Ag.

    Science.gov (United States)

    Sariyer, Ilker Kudret; Merabova, Nana; Patel, Prem Kumer; Knezevic, Tijana; Rosati, Alessandra; Turco, Maria C; Khalili, Kamel

    2012-01-01

    JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.

  8. Localization and Expression of the Proto-Oncoprotein BRX in the Mouse Brain and Pituitary

    National Research Council Canada - National Science Library

    Eddington, David

    2003-01-01

    .... Results indicated that Brx is expressed in specific regions of the brain and pituitary. Furthermore, the results indicate that differences exist in both brain and pituitary tissue of male and female mice with greater expression in the female...

  9. Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5–based Constructs

    Science.gov (United States)

    Alonso-Padilla, Julio; Papp, Tibor; Kaján, Győző L; Benkő, Mária; Havenga, Menzo; Lemckert, Angelique; Harrach, Balázs; Baker, Andrew H

    2016-01-01

    Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes. PMID:26478249

  10. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of

  11. Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

    Science.gov (United States)

    Yang, Teng-Chieh; Maluf, Nasib Karl

    2012-02-21

    Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

  12. In-vitro evaluation of ion-exchange microspheres for the sustained release of liposomal-adenoviral conjugates.

    Science.gov (United States)

    Steel, Jason C; Cavanagh, Heather M A; Burton, Mark A; Dingwall, Daniel; Kalle, Wouter H J

    2004-03-24

    This study looks at the development of a novel combination vector consisting of adenovirus conjugated to liposomes (AL complexes) bound to cation-exchanging microspheres (MAL complexes). With adenovirus having a net negative charge and the liposomes a net positive charge it was possible to modify the net charge of the AL complexes by varying the concentrations of adenovirus to liposomes. The modification of the net charge resulted in altered binding and release characteristics. Of the complexes tested, the 5:1 and 2:1 ratio AL complexes were able to be efficiently bound by the microspheres and exhibited sustained release over 24 h. The 1:1 and 1:2 AL complexes, however, bound poorly to the microspheres and were rapidly released. In addition the MAL complexes also were able to reduce the toxicity of the AL complexes, which was seen with the 10:1 ratio. The AL complexes showed considerably more toxicity alone than in combination with microspheres, highlighting a potential benefit of this vector.

  13. Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Anitra L Farrow

    2014-08-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5 vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83. This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease.

  14. An adenoviral vector expressing lipoprotein A, a major antigen of Mycoplasma mycoides subspecies mycoides, elicits robust immune responses in mice.

    Science.gov (United States)

    Carozza, Marlène; Rodrigues, Valérie; Unterfinger, Yves; Galea, Sandra; Coulpier, Muriel; Klonjkowski, Bernard; Thiaucourt, François; Totté, Philippe; Richardson, Jennifer

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Immunization With AFP + GM CSF Plasmid Prime and AFP Adenoviral Vector Boost in Patients With Hepatocellular Carcinoma

    Science.gov (United States)

    2015-12-01

    Hepatocellular Carcinoma; Hepatoma; Liver Cancer, Adult; Liver Cell Carcinoma; Liver Cell Carcinoma, Adult; Cancer of Liver; Cancer of the Liver; Cancer, Hepatocellular; Hepatic Cancer; Hepatic Neoplasms; Hepatocellular Cancer; Liver Cancer; Neoplasms, Hepatic; Neoplasms, Liver

  16. An anti-VEGF ribozyme embedded within the adenoviral VAI sequence inhibits glioblastoma cell angiogenic potential in vitro.

    Science.gov (United States)

    Ciafrè, Silvia Anna; Niola, Francesco; Wannenes, Francesca; Farace, Maria Giulia

    2004-01-01

    Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis, where it functions as one of the major angiogenic factors sustaining growth and draining catabolites. In this study, we developed an anti-VEGF ribozyme targeted to the 5' part of human VEGF mRNA. We endowed this ribozyme with an additional feature expected to improve its activity in vivo, by cloning it into a VAI transcriptional cassette. VAI is originally part of the adenovirus genome, and is characterized by high transcription rates, good stability due to its strong secondary structure and cytoplasmic localization. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secreted in the extracellular medium, indicating a good biological activity of the ribozyme. Moreover, this reduction in VEGF secretion had the important functional consequence of drastically diminishing the formation of tube-like structures of human umbilical vascular endothelial cells in a Matrigel in vitro angiogenesis assay. In conclusion, our VAI-embedded anti-VEGF ribozyme is a good inhibitor of angiogenesis in vitro, in a glioblastoma cell context. Thus, it may represent a useful tool for future applications in vivo, for antiangiogenic gene therapy of glioblastoma and of highly vascularized tumors. Copyright 2004 S. Karger AG, Basel

  17. Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitro

    NARCIS (Netherlands)

    Kraaij, R; van Rijswijk, ALCT; Haisma, HJ; Bangma, CH

    2005-01-01

    BACKGROUND. Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and, expression of CAR may be

  18. Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms.

    Directory of Open Access Journals (Sweden)

    Helen N Jones

    Full Text Available Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor -1 (hIGF-1 in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined glucose transporter expression and localization in both a mouse model of IUGR and a model of human trophoblast, the BeWo Choriocarcinoma cell line.At gestational day 18, animals were divided into four groups; sham-operated controls, uterine artery branch ligation (UABL, UABL+Ad-hIGF-1 (10(8 PFU, UABL+Ad-LacZ (10(8 PFU. At gestational day 20, pups and placentas were harvested by C-section. For human studies, BeWo choriocarcinoma cells were grown in F12 complete medium +10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 hours. MOIs of 10∶1 and 100∶1 were utilized. The RNA, protein expression and localization of glucose transporters GLUT1, 3, 8, and 9 were analyzed by RT-PCR, Western blot and immunohistochemistry.In both the mouse placenta and BeWo, GLUT1 regulation was linked to altered protein localization. GLUT3, localized to the mouse fetal endothelial cells, was reduced in placental insufficiency but maintained with Ad-I GF-1 treatment. Interestingly, GLUT8 expression was reduced in the UABL placenta but up-regulated following Ad-IGF-1 in both mouse and human systems. GLUT9 expression in the mouse was increased by Ad-IGF-1 but this was not reflected in the BeWo, where Ad-IGF-1 caused moderate membrane relocalization.Enhanced GLUT isoform transporter expression and relocalization to the membrane may be an important mechanism in Ad-hIGF-1mediated correction of placental insufficiency.

  19. The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

    Science.gov (United States)

    Hale, T K; Braithwaite, A W

    1999-08-20

    Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

  20. Differential Mechanisms for SHP2 Binding and Activation Are Exploited by Geographically Distinct Helicobacter pylori CagA Oncoproteins

    Directory of Open Access Journals (Sweden)

    Takeru Hayashi

    2017-09-01

    Full Text Available Helicobacter pylori East Asian CagA is more closely associated with gastric cancer than Western CagA. Here we show that, upon tyrosine phosphorylation, the East Asian CagA-specific EPIYA-D segment binds to the N-SH2 domain of pro-oncogenic SHP2 phosphatase two orders of magnitude greater than Western CagA-specific EPIYA-C. This high-affinity binding is achieved via cryptic interaction between Phe at the +5 position from phosphotyrosine in EPIYA-D and a hollow on the N-SH2 phosphopeptide-binding floor. Also, duplication of EPIYA-C in Western CagA, which increases gastric cancer risk, enables divalent high-affinity binding with SHP2 via N-SH2 and C-SH2. These strong CagA bindings enforce enzymatic activation of SHP2, which endows cells with neoplastic traits. Mechanistically, N-SH2 in SHP2 is in an equilibrium between stimulatory “relaxed” and inhibitory “squeezed” states, which is fixed upon high-affinity CagA binding to the “relaxed” state that stimulates SHP2. Accordingly, East Asian CagA and Western CagA exploit distinct mechanisms for SHP2 deregulation.

  1. The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

    International Nuclear Information System (INIS)

    Zhang Weifang; Li Jing; Kanginakudru, Sriramana; Zhao Weiming; Yu Xiuping; Chen, Jason J.

    2010-01-01

    HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

  2. Induction of Epstein-Barr Virus Oncoprotein LMP1 by Transcription Factors AP-2 and Early B Cell Factor

    Science.gov (United States)

    Noda, Chieko; Narita, Yohei; Watanabe, Takahiro; Yoshida, Masahiro; Ashio, Keiji; Sato, Yoshitaka; Goshima, Fumi; Kanda, Teru; Yoshiyama, Hironori; Tsurumi, Tatsuya; Kimura, Hiroshi

    2016-01-01

    ABSTRACT Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-Jκ, NF-κB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1. PMID:26819314

  3. Prognostic Significance of c-erbB-2 (HER-2/neu) Oncoprotein in Benign and Malignant Breast Tumor Tissue

    Czech Academy of Sciences Publication Activity Database

    Šimíčková, M.; Pecen, Ladislav; Černoch, M.; Vagunda, V.; Pochmon, D.; Vermousek, I.; Lang, B.; Pačovský, Z.

    1999-01-01

    Roč. 14, č. 4 (1999), s. 13-22 ISSN 0886-3849 Grant - others:IGA MZ(CZ) IZ2924 Institutional research plan: AV0Z1030915 Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 0.326, year: 1999

  4. Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability

    NARCIS (Netherlands)

    D.J. White (Daniel); R.D. Unwin (Richard); E.M.J. Bindels (Eric); A. Pierce (Andrew); H.Y. Teng (Hsiang-Ying); J. Muter (Joanne); B. Greystoke (Brigit); T.D. Somerville (Tim); D.J. Griffiths (Derek); S. Lovell (Simon); T.C.P. Somervaille (Tim); H.R. Delwel (Ruud); A.D. Whetton (Anthony); S. Meyer (Stefan)

    2013-01-01

    textabstractThe EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1

  5. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

    Directory of Open Access Journals (Sweden)

    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  6. Production of human papillomavirus type16 E7 oncoprotein fused with ß-glucuronidase in transgenic tomato and potato

    Czech Academy of Sciences Publication Activity Database

    Bříza, Jindřich; Pavingerová, Daniela; Vlasák, Josef; Ludvíková, V.; Niedermeierová, Hana

    2007-01-01

    Roč. 51, č. 2 (2007), s. 268-276 ISSN 0006-3134 R&D Projects: GA ČR GA521/05/2092 Institutional research plan: CEZ:AV0Z50510513 Keywords : transgenic plants * human papillomavirus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.259, year: 2007

  7. Requirement of the coiled-coil domain of PML-RARα oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation

    International Nuclear Information System (INIS)

    Kim, Young-Eui; Kim, Dong-Yeon; Lee, Jang-Mi; Kim, Seong-Tae; Han, Tae-Hee; Ahn, Jin-Hyun

    2005-01-01

    Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(ΔC-C)-RARα, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the ΔC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the ΔC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the ΔC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARα. However, the RA-mediated induction of C/EBPβ in cells expressing the ΔC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARα

  8. The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

    International Nuclear Information System (INIS)

    Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F.; Poumay, Yves

    2007-01-01

    Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity

  9. Autophagy collaborates with ubiquitination to downregulate oncoprotein E2A/Pbx1 in B-cell acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Yuan, N; Song, L; Lin, W; Cao, Y; Xu, F; Liu, S; Zhang, A; Wang, Z; Li, X; Fang, Y; Zhang, H; Zhao, W; Hu, S; Wang, J; Zhang, S

    2015-01-01

    B-cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. We present here that autophagy is downregulated in pediatric B-ALL, suggesting a possible link between autophagy failure and pediatric B-ALL leukemogenesis. With a pediatric t(1;19) B-ALL xenograft mouse model, we show here that activation of autophagy by preventive administration of rapamycin improved the survival of leukemia animals by partial restoration of hematopoietic stem/progenitor cells, whereas treatment of the animals with rapamycin caused leukemia bone marrow cell-cycle arrest. Activation of autophagy in vitro or in vivo by rapamycin or starvation downregulated oncogenic fusion protein E2A/Pbx1. Furthermore, E2A/Pbx1 was found to be colocalized with autophagy marker LC3 in autolysosomes and with ubiquitin in response to autophagy stimuli, whereas autophagy or ubiquitination inhibitor blocked these colocalizations. Together, our data suggest a collaborative action between autophagy and ubiquitination in the degradation of E2A/Pbx1, thereby revealing a novel strategy for targeted preventive or treatment therapy on the pediatric ALL

  10. Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

    Science.gov (United States)

    Richards, Mark W; O'Regan, Laura; Roth, Daniel; Montgomery, Jessica M; Straube, Anne; Fry, Andrew M; Bayliss, Richard

    2015-05-01

    Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

  11. Positive and negative regulation of cell proliferation by E2F-1: influence of protein level and human papillomavirus oncoproteins

    DEFF Research Database (Denmark)

    Melillo, R M; Helin, K; Lowy, D R

    1994-01-01

    E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High...... to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1...... in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7...

  12. Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells

    International Nuclear Information System (INIS)

    Lagrange, Magali; Boulade-Ladame, Charlotte; Mailly, Laurent; Weiss, Etienne; Orfanoudakis, Georges; Deryckere, Francois

    2007-01-01

    The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies

  13. Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis, E-mail: l-laimins@northwestern.edu

    2014-03-15

    Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.

  14. A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles▿

    OpenAIRE

    Chung, Sang-Hyuk; Frese, Kristopher K.; Weiss, Robert S.; Prasad, B. V. Venkataram; Javier, Ronald T.

    2007-01-01

    Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, inc...

  15. The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression

    Science.gov (United States)

    He, Chunbo; Mao, Dagan; Hua, Guohua; Lv, Xiangmin; Chen, Xingcheng; Angeletti, Peter C; Dong, Jixin; Remmenga, Steven W; Rodabaugh, Kerry J; Zhou, Jin; Lambert, Paul F; Yang, Peixin; Davis, John S; Wang, Cheng

    2015-01-01

    The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes-associated protein (YAP). Here, we show that YAP plays a central role in controlling the progression of cervical cancer. Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer. TGF-α and amphiregulin (AREG), via EGFR, inhibit the Hippo signaling pathway and activate YAP to induce cervical cancer cell proliferation and migration. Activated YAP allows for up-regulation of TGF-α, AREG, and EGFR, forming a positive signaling loop to drive cervical cancer cell proliferation. HPV E6 protein, a major etiological molecule of cervical cancer, maintains high YAP protein levels in cervical cancer cells by preventing proteasome-dependent YAP degradation to drive cervical cancer cell proliferation. Results from human cervical cancer genomic databases and an accepted transgenic mouse model strongly support the clinical relevance of the discovered feed-forward signaling loop. Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer. PMID:26417066

  16. Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1

    International Nuclear Information System (INIS)

    Wierstra, Inken; Alves, Juergen

    2008-01-01

    FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27

  17. Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

    Science.gov (United States)

    Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

    2013-10-17

    Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

  18. Assessing Protection Against OP Pesticides and Nerve Agents Provided by Wild-Type HuPON1 Purified from Trichoplusia ni Larvae or Induced via Adenoviral Infection

    Science.gov (United States)

    2013-01-01

    by tracking the production of p-nitrophenol (at A412) from paraoxon (9 mM in 50 mM Tris, 10 mM CaCl2, pH 7.4), as previously described [24]. To...poisoning ( salivation and convulsive chewing) that resolved within 1 h of OP exposure, while the remaining animal died between 8 and 16 h after

  19. E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

    Science.gov (United States)

    Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

    1999-05-01

    Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

  20. Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

    International Nuclear Information System (INIS)

    Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

    2004-01-01

    Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

  1. Measurement of feline cytokines interleukin-12 and interferon- g produced by heat inducible gene therapy adenoviral vector using real time PCR

    International Nuclear Information System (INIS)

    Siddiqui, F.; Avery, P.R.; Ullrich, R.L.; LaRue, S.M.; Dewhirst, M.W.; Li, C.-Y.

    2003-01-01

    Biologic tumor therapy using Interleukin-12 (IL-12) has shown promise as an adjuvant to radiation therapy. The goals for cancer gene immunotherapy include effective eradication of established tumors and generation of a lasting systemic immune response. Among the cytokines, IL-12 has been found to be most effective gene in eradicating experimental tumors, preventing the development of metastases, and eliciting long-term antitumor immunity. Depending on the tumor model, IL-12 can exert antitumor activities via T cells, NK cells or NKT cells. It induces the production of IFN-g and IFN-inducible protein-10. It is also postulated to have antiangiogenic effects, thus inhibiting tumor formation and metastases. However, its use in clinical trials has been restricted largely owing to its systemic hematologic and hepatotoxicity. We tested the efficacy of adenovirus mediated expression of feline IL-12 gene placed under the control of an inducible promoter, the heat shock proteins (hsp70B). This places gene expression under the control of an external physical agent (hyperthermia), thus offering an 'on-off' switch and potentially reducing systemic toxicity by restricting its expression locally to the tumor. Crandell Feline Kidney (CrFK) cells were infected using the construct and the supernatant was then used to stimulate production of interferon g (IFN-g) in feline peripheral blood mononuclear cells (PBMC). As there is no commercially available ELISA kit currently available to detect or measure feline cytokines, we used real time-PCR to measure cytokine mRNA. These results will be used to initiate a clinical trial in cats with soft tissue sarcomas examining hyperthermia Induced gene therapy in conjunction with radiation therapy. The real time- PCR techniques developed here will be used to quantitatively measure cytokine mRNA levels in the punch biopsy samples obtained from the cats during the clinical trial. Support for this study was in part by NCI grant CA72745

  2. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Kim, Sung Jin; Lee, Heui Ran [Medical Research Center, Seoul National University, Seoul (Korea, Republic of)

    2008-10-15

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1{+-}4.7%, 54.0{+-}6.4%, 85.7{+-}8.7%, and 98.4{+-}1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704{+-}6,659 picomole/10{sup 6} cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168{+-}2,134 picomole/10{sup 6} cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

  3. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    NARCIS (Netherlands)

    Yang, X.; Kraan, P.M. van der; Dolder, J. van den; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2007-01-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and

  4. Replication and virus-induced transcriptome of HAdV-5 in normal host cells versus cancer cells--differences of relevance for adenoviral oncolysis.

    Directory of Open Access Journals (Sweden)

    Dominik E Dorer

    Full Text Available Adenoviruses (Ads, especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication.

  5. Adenoviral vector-mediated expression of a foreign gene in peripheral nerve tissue bridges implanted in the injured peripheral and central nervous system

    NARCIS (Netherlands)

    Blits, B; Dijkhuizen, Paul A; Carlstedt, Thomas P; Poldervaart, H A; Schiemanck, S; Boer, G J; Verhaagen, J

    1999-01-01

    Axons of the CNS do normally not regenerate after injury, in contrast to axons of the PNS. This is due to a different microenvironment at the site of the lesion as well as a particular intrinsic program of axonal regrowth. Although transplantation of peripheral nerve tissue bridges is perhaps the

  6. Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

    Directory of Open Access Journals (Sweden)

    Amita Joshi

    Full Text Available Treatment of invasive adenovirus (Ad disease in hematopoietic stem cell transplant (SCT recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977 and late protein hexon (H-892 were compared in peripheral blood (PB and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

  7. Enhanced Differentiation of Three-Gene-Reprogrammed Induced Pluripotent Stem Cells into Adipocytes via Adenoviral-Mediated PGC-1α Overexpression

    Directory of Open Access Journals (Sweden)

    Yi-Jen Chen

    2011-11-01

    Full Text Available Induced pluripotent stem cells formed by the introduction of only three factors, Oct4/Sox2/Klf4 (3-gene iPSCs, may provide a safer option for stem cell-based therapy than iPSCs conventionally introduced with four-gene iPSCs. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α plays an important role during brown fat development. However, the potential roles of PGC-1α in regulating mitochondrial biogenesis and the differentiation of iPSCs are still unclear. Here, we investigated the effects of adenovirus-mediated PGC-1α overexpression in 3-gene iPSCs. PGC-1α overexpression resulted in increased mitochondrial mass, reactive oxygen species production, and oxygen consumption. Microarray-based bioinformatics showed that the gene expression pattern of PGC-1α-overexpressing 3-gene iPSCs resembled the expression pattern observed in adipocytes. Furthermore, PGC-1α overexpression enhanced adipogenic differentiation and the expression of several brown fat markers, including uncoupling protein-1, cytochrome C, and nuclear respiratory factor-1, whereas it inhibited the expression of the white fat marker uncoupling protein-2. Furthermore, PGC-1α overexpression significantly suppressed osteogenic differentiation. These data demonstrate that PGC-1α directs the differentiation of 3-gene iPSCs into adipocyte-like cells with features of brown fat cells. This may provide a therapeutic strategy for the treatment of mitochondrial disorders and obesity.

  8. Safety and immunogenicity of an HIV adenoviral vector boost after DNA plasmid vaccine prime by route of administration: a randomized clinical trial.

    Directory of Open Access Journals (Sweden)

    Beryl A Koblin

    Full Text Available In the development of HIV vaccines, improving immunogenicity while maintaining safety is critical. Route of administration can be an important factor.This multicenter, open-label, randomized trial, HVTN 069, compared routes of administration on safety and immunogenicity of a DNA vaccine prime given intramuscularly at 0, 1 and 2 months and a recombinant replication-defective adenovirus type 5 (rAd5 vaccine boost given at 6 months by intramuscular (IM, intradermal (ID, or subcutaneous (SC route. Randomization was computer-generated by a central data management center; participants and staff were not blinded to group assignment. The outcomes were vaccine reactogenicity and humoral and cellular immunogenicity. Ninety healthy, HIV-1 uninfected adults in the US and Peru, aged 18-50 were enrolled and randomized. Due to the results of the Step Study, injections with rAd5 vaccine were halted; thus 61 received the booster dose of rAd5 vaccine (IM: 20; ID:21; SC:20. After the rAd5 boost, significant differences by study arm were found in severity of headache, pain and erythema/induration. Immune responses (binding and neutralizing antibodies, IFN-γ ELISpot HIV-specific responses and CD4+ and CD8+ T-cell responses by ICS at four weeks after the rAd5 booster were not significantly different by administration route of the rAd5 vaccine boost (Binding antibody responses: IM: 66.7%; ID: 70.0%; SC: 77.8%; neutralizing antibody responses: IM: 11.1%; ID: 0.0%; SC 16.7%; ELISpot responses: IM: 46.7%; ID: 35.3%; SC: 44.4%; CD4+ T-cell responses: IM: 29.4%; ID: 20.0%; SC: 35.3%; CD8+ T-cell responses: IM: 29.4%; ID: 16.7%; SC: 50.0%.This study was limited by the reduced sample size. The higher frequency of local reactions after ID and SC administration and the lack of sufficient evidence to show that there were any differences in immunogenicity by route of administration do not support changing route of administration for the rAd5 boost.ClinicalTrials.gov NCT00384787.

  9. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    International Nuclear Information System (INIS)

    Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun; Kim, Sung Jin; Lee, Heui Ran

    2008-01-01

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1±4.7%, 54.0±6.4%, 85.7±8.7%, and 98.4±1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704±6,659 picomole/10 6 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168±2,134 picomole/10 6 cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression

  10. Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

    International Nuclear Information System (INIS)

    Doloff, Joshua C; Su, Ting; Waxman, David J

    2010-01-01

    Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 'factory' cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products

  11. Adenoviral vaccination combined with CD40 stimulation and CTLA-4 blockage can lead to complete tumor regression in a murine melanoma model

    DEFF Research Database (Denmark)

    Sørensen, Maria Rathmann; Holst, Peter J; Steffensen, Maria Abildgaard

    2010-01-01

    that the delay in tumor growth can be converted to complete regression and long-term survival in 30-40% of the mice by a booster vaccination plus combinational treatment with agonistic anti-CD40 monoclonal antibodies (mAb) and anti-CTLA-4 mAb. Regarding the mechanism underlying the improved clinical effect......, analysis of the tumor-specific response revealed a significantly prolonged tumor-specific CD8 T cell response in spleens of the mice receiving the combinational treatment compared with mice receiving either treatment individually. Matching this, CD8 T cell depletion completely prevented tumor control...

  12. Comparison of human sodium iodide symporter (hNIS) gene expression between lentiviral and adenoviral vectors in rat mesenchymal stem cell

    International Nuclear Information System (INIS)

    Park, So Yeon; Lee, Won Woo; Kim, Sung Jin; Lee, Heui Ran; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun

    2007-01-01

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been done. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated stably hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning the hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and Rad-hNIS transduced rMSC (adeno-hNIS-rMSC) was evaluated for the hNIS expression 48 hours post infection at MOI 1, 5, 20, 50, and 100. The hNIS expression in lenti-hNIS-rMSC or adeno-hNIS-rMSC was assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry using mono-clonal anti-hNIS antibody revealed that intensity of hNIS immunoreactivity in lenti-hNIS-rMSC was greater than that in adeno-hNIS-rMSC at MOl 20 but lower than that at MOl 50. Western blot analysis also showed that lenti-hNIS-rMSC was intermediate between adeno-hNIS-rMSCs at MOl 20 and 50 in hNIS expression. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (297046659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (61682134 picomole/106 cells). These results suggest that lentivirus mediated hNIS expression is greater in terms of hNIS function but lower in terms of hNIS protein amount than adenovirus mediated hNIS expression 48 hours post infection. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative viral efficiency of transgene expression

  13. Combining Cell Type-Restricted Adenoviral Targeting with Immunostaining and Flow Cytometry to Identify Cells-of-Origin of Lung Cancer.

    Science.gov (United States)

    Best, Sarah A; Kersbergen, Ariena; Asselin-Labat, Marie-Liesse; Sutherland, Kate D

    2018-01-01

    Lung cancers display considerable intertumoral heterogeneity, leading to the classification of distinct tumor subtypes. Our understanding of the genetic aberrations that underlie tumor subtypes has been greatly enhanced by recent genomic sequencing studies and state-of-the-art gene targeting technologies, highlighting evidence that distinct lung cancer subtypes may be derived from different "cells-of-origin". Here, we describe the intra-tracheal delivery of cell type-restricted Ad5-Cre viruses into the lungs of adult mice, combined with immunohistochemical and flow cytometry strategies for the detection of lung cancer-initiating cells in vivo.

  14. Preclinical safety, toxicology, and biodistribution study of adenoviral gene therapy with sVEGFR-2 and sVEGFR-3 combined with chemotherapy for ovarian cancer.

    Science.gov (United States)

    Tuppurainen, Laura; Sallinen, Hanna; Kokki, Emmi; Koponen, Jonna; Anttila, Maarit; Pulkkinen, Kati; Heikura, Tommi; Toivanen, Pyry; Hämäläinen, Kirsi; Kosma, Veli-Matti; Heinonen, Seppo; Alitalo, Kari; Ylä-Herttuala, Seppo

    2013-03-01

    Abstract Antiangiogenic and antilymphangiogenic gene therapy with soluble vascular endothelial growth factor receptor-2 (VEGFR-2) and soluble VEGFR-3 in combination with chemotherapy is a potential new treatment for ovarian carcinoma. We evaluated the safety, toxicology, and biodistribution of intravenous AdsVEGFR-2 and AdsVEGFR-3 combined with chemotherapy in healthy rats (n=90) before entering a clinical setting. The study groups were: AdLacZ and AdLacZ with chemotherapy as control groups, low dose AdsVEGFR-2 and AdsVEGFR-3, high dose AdsVEGFR-2 and AdsVEGFR-3, combination of low dose AdsVEGFR-2 and AdsVEGFR-3 with chemotherapy, combination of high dose AdsVEGFR-2 and AdVEGFR-3 with chemotherapy, and chemotherapy only. The follow-up time was 4 weeks. Safety and toxicology were assessed by monitoring the clinical status of the animals and by histological, hematological, and clinical chemistry parameters. For the biodistribution studies, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used. Low dose (2×10(10) vp) AdsVEGFR-2 and AdsVEGFR-3 gene therapy was well tolerated, even when gene therapy was combined with chemotherapy. Notably, only transient elevation of liver enzymes and mild regenerative changes were seen in liver after the gene transfer in the groups that received high doses (2×10(11) vp) of AdsVEGFR-2 and AdsVEGFR-3 with or without chemotherapy. No life-threatening adverse effects were noticed in any of the treatment groups. The highest protein concentration of soluble VEGFR-2 (sVEGFR-2) in circulation was seen 1 week after the gene transfer. The combination of chemotherapy to gene therapy seemed to prolong the time of detectable transgene protein at least 1 week in the circulation. The expression of AdsVEGFR-2 and AdsVEGFR-3 transgenes was mainly seen in the liver and spleen as detected by qRT-PCR. According to these results, AdsVEGFR-2 and AdsVEGFR-3 gene therapy combined with chemotherapy is safe and can be brought to clinical testing in ovarian cancer patients.

  15. Biotherapeutics as alternatives to antibiotics: effects of adenoviral delivered IFN-alpha and G-CSF on innate and adaptive immunity in swine

    Science.gov (United States)

    Acceptable alternatives to the use of antibiotics in swine practice need to be explored. The use of immunomodulators is a promising area for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease during periods of peak disease incidence. We developed a method to ci...

  16. Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy.

    Science.gov (United States)

    Liu, Sheng-Hung; Lin, Chao-Hsiung; Liang, Fong-Ping; Chen, Pei-Fen; Kuo, Cheng-Deng; Alam, Mohd Mujahid; Maiti, Barnali; Hung, Shih-Kai; Chi, Chin-Wen; Sun, Chung-Ming; Fu, Shu-Ling

    2014-01-15

    Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Hoffmeisterová, Hana; Plchová, Helena; Synková, Helena; Poláková, I.; Dušková, M.; Šmahel, M.

    2013-01-01

    Roč. 113, č. 1 (2013), s. 81-90 ISSN 0167-6857 R&D Projects: GA ČR GA521/09/1525; GA ČR(CZ) GAP501/12/1761 Institutional research plan: CEZ:AV0Z50380511 Keywords : Transient expression * Molecular farming * Potato virus X Subject RIV: EE - Microbiology, Virology Impact factor: 2.612, year: 2013

  18. The Putative PAX8/PPARγ Fusion Oncoprotein Exhibits Partial Tumor Suppressor Activity through Up-Regulation of Micro-RNA-122 and Dominant-Negative PPARγ Activity.

    Science.gov (United States)

    Reddi, Honey V; Madde, Pranathi; Milosevic, Dragana; Hackbarth, Jennifer S; Algeciras-Schimnich, Alicia; McIver, Bryan; Grebe, Stefan K G; Eberhardt, Norman L

    2011-01-01

    In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

  19. Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

    International Nuclear Information System (INIS)

    Tao Yongguang; Song Xing; Deng Xiyun; Xie Daxin; Lee, Leo M.; Liu Yiping; Li Wei; Li Lili; Deng Lin; Wu Qiao; Gong Jianping; Cao Ya

    2005-01-01

    Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma

  20. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    International Nuclear Information System (INIS)

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit; Kundu, Chanakya N.; Verma, Subhash C.; Choudhuri, Tathagata

    2014-01-01

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways

  1. CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

    Science.gov (United States)

    Cheong, Jit Kong; Gunaratnam, Lakshman; Hsu, Stephen I-Hong

    2008-04-25

    Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

  2. Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours

    Directory of Open Access Journals (Sweden)

    Miller William R

    2005-09-01

    Full Text Available Abstract Background The tissue-specific translation elongation factor eEF1A2 was recently shown to be a potential oncogene that is overexpressed in ovarian cancer. Although there is no direct evidence for an involvement of eEF1A2 in breast cancer, the genomic region to which EEF1A2 maps, 20q13, is frequently amplified in breast tumours. We therefore sought to establish whether eEF1A2 expression might be upregulated in breast cancer. Methods eEF1A2 is highly similar (98% to the near-ubiquitously expressed eEF1A1 (formerly known as EF1-α making analysis with commercial antibodies difficult. We have developed specific anti-eEF1A2 antibodies and used them in immunohistochemical analyses of tumour samples. We report the novel finding that although eEF1A2 is barely detectable in normal breast it is moderately to strongly expressed in two-thirds of breast tumours. This overexpression is strongly associated with estrogen receptor positivity. Conclusion eEF1A2 should be considered as a putative oncogene in breast cancer that may be a useful diagnostic marker and therapeutic target for a high proportion of breast tumours. The oncogenicity of eEF1A2 may be related to its role in protein synthesis or to its potential non-canonical functions in cytoskeletal remodelling or apoptosis.

  3. The oncoprotein v-Myb activates transcription of Gremlin 2 during in vitro differentiation of the chicken neural crest to melanoblasts

    Czech Academy of Sciences Publication Activity Database

    Starostová, Michaela; Čermák, Vladimír; Dvořáková, Marta; Karafiát, Vít; Kosla, Jan; Dvořák, Michal

    2014-01-01

    Roč. 540, č. 1 (2014), s. 122-129 ISSN 0378-1119 R&D Projects: GA AV ČR KAN200520801 Institutional support: RVO:68378050 Keywords : Melanocyte development * Tamoxifen-inducible v-Myb * v-Myb-dependent genes * PRDC * KRT19 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.138, year: 2014

  4. A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Hansen, Christina N; Vinther, Jeppe

    2003-01-01

    RNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic m...... from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream...... of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells....

  5. MHC class I+ and class I(-)HPV16-associated tumours expressing the E7 oncoprotein do not cross-react in immunization/challenge experiments

    Czech Academy of Sciences Publication Activity Database

    Šímová, Jana; Mikyšková, Romana; Vonka, V.; Bieblová, Jana; Bubeník, Jan; Jandlová, Táňa

    2003-01-01

    Roč. 2003, č. 49 (2003), s. 230-234 ISSN 0015-5500 Institutional research plan: CEZ:AV0Z5052915 Keywords : HPV 16 * MHC class I expression * tumour vaccines Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.527, year: 2003

  6. Nuclear import of high risk HPV16 E7 oncoprotein is mediated by its zinc-binding domain via hydrophobic interactions with Nup62

    Energy Technology Data Exchange (ETDEWEB)

    Eberhard, Jeremy; Onder, Zeynep; Moroianu, Junona, E-mail: moroianu@bc.edu

    2013-11-15

    We previously discovered that nuclear import of high risk HPV16 E7 is mediated by a cNLS located within the zinc-binding domain via a pathway that is independent of karyopherins/importins (Angeline et al., 2003; Knapp et al., 2009). In this study we continued our characterization of the cNLS and nuclear import pathway of HPV16 E7. We find that an intact zinc-binding domain is essential for the cNLS function in mediating nuclear import of HPV16 E7. Mutagenesis of cysteine residues to alanine in each of the two CysXXCys motifs involved in zinc-binding changes the nuclear localization of the EGFP-16E7 and 2xEGFP-16E7 mutants. We further discover that a patch of hydrophobic residues, {sub 65}LRLCV{sub 69}, within the zinc-binding domain of HPV16 E7 mediates its nuclear import via hydrophobic interactions with the FG domain of the central channel nucleoporin Nup62. - Highlights: • An intact zinc-binding domain is essential for the nuclear localization of HPV16 E7. • Identification of a hydrophobic patch that is critical for the nuclear import of HPV16 E7. • HPV16 E7 interacts via its zinc-binding domain with the FG domain of Nup62.

  7. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit [Division of Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023 (India); Kundu, Chanakya N. [School of Biotechnology, KIIT University, Bhubaneswar (India); Verma, Subhash C. [Department of Microbiology and Immunology, University of Nevada, School of Medicine, Reno, NV 89557 (United States); Choudhuri, Tathagata, E-mail: tatha@ils.res.in [Division of Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023 (India); Department of Biotechnology, Siksha Bhavana, Visva Bharati, Santiniketan, Bolpur (India)

    2014-01-05

    The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways.

  8. Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

    Directory of Open Access Journals (Sweden)

    Janin Chandra

    Full Text Available Antigen presenting cells (APCs in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.

  9. HTLV-2B Tax oncoprotein is modified by ubiquitination and sumoylation and displays intracellular localization similar to its homologue HTLV-1 Tax

    International Nuclear Information System (INIS)

    Turci, Marco; Lodewick, Julie; Righi, Paola; Polania, Angela; Romanelli, Maria Grazia; Bex, Francoise; Bertazzoni, Umberto

    2009-01-01

    HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties of their individual transactivating Tax proteins. By using internal Flag-6His tagged Tax-1 and Tax-2B, which display transcriptional activities comparable to the untagged proteins and can be recognized by a single anti-Flag antibody, we demonstrate that Tax-2B is modified by ubiquitination and sumoylation. In addition, Tax2B is distributed in punctuate nuclear structures that include the RelA subunit of NF-κB, as has been previously demonstrated for Tax-1

  10. Radioimmunotherapy with an antibody to HPV16 E6 oncoprotein is effective in experimental cervical tumor expressing low levels of E6

    Science.gov (United States)

    Jiang, Zewei; Wang, Xing Guo; Einstein, Mark H; Goldberg, Gary L; Casadevall, Arturo

    2010-01-01

    Purpose HPV16 is associated with ∼50% of all cervical cancers worldwide. The E6 and E7 genes of oncogenic HPV types, such as HPV16, are necessary for the HPV transforming function and tumorogenesis making them ideal targets for novel treatments. Radioimmunotherapy employs systemically administered radiolabeled monoclonal antibodies (mAbs) that bind to tumor-associated antigens. Previously we demonstrated in mice that radioimmunotherapy targeting viral antigens with mAb to HPV16 E6 suppressed CasKi cervical tumors expressing high levels of E6 (∼600 copies of HPV per cell). However, that study opened the question whether radioimmunotherapy can suppress the growth of cervical tumors with low E6 and E7 expression, such as may be seen in patients. Experimental Design We evaluated the expression of E6 in patients' tumors and in the SiHa cell line expressing low levels of E6 and E7 (1–2 copies of HPV per cell) and found them comparable. We initiated SiHa tumors in nude mice, radiolabeled C1P5 mAb to E6 with a beta-emitter 188-Rhenium (188Re) and treated tumor-bearing mice with: (1) 200 µCi 188Re-C1P5 alone; (2) proteasome inhibitor MG132 alone; (3) MG132 followed by 200 µCi 188Re-C1P5; (4) unlabeled C1P5; (5) 200 µCi 188Re-18B7 (isotype-matching control mAb); (6) no treatment. 188Re-C1P5 alone and in combination with MG-132 significantly retarded tumor growth compared to all control groups. Conclusions Our data demonstrate the possibility to suppress tumor growth by targeting viral antigens even in cervical tumors with low E6 expression and provide additional evidence for the potential usefulness of radioimmunotherapy targeting HPV-related antigens in the clinic. PMID:20861673

  11. Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours

    International Nuclear Information System (INIS)

    Tomlinson, Victoria AL; Newbery, Helen J; Wray, Naomi R; Jackson, Juliette; Larionov, Alexey; Miller, William R; Dixon, J Michael; Abbott, Catherine M

    2005-01-01

    The tissue-specific translation elongation factor eEF1A2 was recently shown to be a potential oncogene that is overexpressed in ovarian cancer. Although there is no direct evidence for an involvement of eEF1A2 in breast cancer, the genomic region to which EEF1A2 maps, 20q13, is frequently amplified in breast tumours. We therefore sought to establish whether eEF1A2 expression might be upregulated in breast cancer. eEF1A2 is highly similar (98%) to the near-ubiquitously expressed eEF1A1 (formerly known as EF1-α) making analysis with commercial antibodies difficult. We have developed specific anti-eEF1A2 antibodies and used them in immunohistochemical analyses of tumour samples. We report the novel finding that although eEF1A2 is barely detectable in normal breast it is moderately to strongly expressed in two-thirds of breast tumours. This overexpression is strongly associated with estrogen receptor positivity. eEF1A2 should be considered as a putative oncogene in breast cancer that may be a useful diagnostic marker and therapeutic target for a high proportion of breast tumours. The oncogenicity of eEF1A2 may be related to its role in protein synthesis or to its potential non-canonical functions in cytoskeletal remodelling or apoptosis

  12. The oncoprotein and stem cell renewal factor BMI1 associates with poor clinical outcome in oesophageal cancer patients undergoing preoperative chemoradiotherapy

    Directory of Open Access Journals (Sweden)

    Yoshikawa Reigetsu

    2012-10-01

    Full Text Available Abstract Background The polycomb group (PcG family BMI1, acting downstream of the hedgehog (Hh pathway, plays an essential role in the self-renewal of haematopoietic, neural, and intestinal stem cells, and is dysregulated in many types of cancer. Our recent report has demonstrated that Hh signalling activation can predict very earlier relapse of oesophageal cancers. As data were not available on the clinical role of BMI1 expression in oesophageal cancers after chemoradiotherapy (CRT, we analysed whether it could be also used to predict disease progression and prognosis in oesophageal cancer patients undergoing trimodality therapy of preoperative CRT and oesophagectomy. Methods Expressions of BMI1 and p16INK4A, a downstream target of PcG, were analysed in 78 patients with histologically confirmed oesophageal squamous cell carcinoma (ESCC after preoperative CRT by immunohistochemical staining. The association of BMI1 and p16INK4A expression with clinicopathologic characteristics was analysed by χ2-test. Survival analysis was carried out by the log-rank test using Kaplan-Meier method. Results Among 78 ESCC patients, 24 patients (30.8% showed BMI1 positivity, mainly localised in the nuclei of tumour cells. Patients harbouring BMI1-positive tumour cells showed significantly poorer prognoses than those without such cells or residual tumours (mean disease-free survival (DFS time 16.8 vs 71.2 months; 3-yr DFS 13.3% vs 49.9%, P=0.002; mean OS time 21.8 vs 76.6 months; 3-yr OS 16.2% vs 54.9%, P=0.0005. There was no significant correlation between p16INK4A expression and BMI1 expression. Conclusions Our study shows that BMI1 expression is a predictor of early relapse and poor prognosis in ESCC after CRT. These findings suggest that BMI1 signal activation might be involved in promoting cancer regrowth and progression after CRT, and might be indicative of emergence of ‘more aggressive’ cancer progenitor cells.

  13. Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Di [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Yuan, Yunsheng [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai (China); Chen, Li [Pharmacy College, Fujian University of Traditional Chinese Medicine, Fuzhou (China); Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Liu, Xin; Belani, Chandra [Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 (United States); Cheng, Hua, E-mail: hcheng@ihv.umaryland.edu [Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Department Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201 (United States)

    2015-08-14

    Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and IκB kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells. - Highlights: • Niclosamide is a promising therapeutic candidate for adult T cell leukemia. • Niclosamide employs a novel mechanism through proteasomal degradation of Tax. • Niclosamide downregulates certain cellular pro-survival molecules.

  14. Enhancement of T cell-mediated and humoral immunity of beta-glucuronidase-based DNA vaccines against HPV16 E7 oncoprotein

    Czech Academy of Sciences Publication Activity Database

    Šmahel, M.; Poláková, I.; Pokorná, D.; Ludvíková, V.; Dušková, M.; Vlasák, Josef

    2008-01-01

    Roč. 33, č. 1 (2008), s. 93-102 ISSN 1019-6439 R&D Projects: GA ČR GA521/05/2092 Institutional research plan: CEZ:AV0Z50510513 Keywords : immunogenicity * anti-E7 vaccines Subject RIV: FD - Oncology ; Hematology Impact factor: 2.234, year: 2008

  15. Enhancement of T cell-mediated and humoral immunity of beta-glucuronidase-based DNA vaccines against HPV16 E7 oncoprotein

    Czech Academy of Sciences Publication Activity Database

    Šmahel, M.; Poláková, I.; Pokorná, D.; Ludvíková, V.; Dušková, M.; Vlasák, Josef

    2008-01-01

    Roč. 16, - (2008), S60 ISSN 0022-1732. [HPV in Human Pathology :International Conference. 01.05.2008-03.05.2008, Prague] R&D Projects: GA ČR GA521/05/2092; GA MZd NR9246 Institutional research plan: CEZ:AV0Z50510513 Keywords : oncology * beta-glucuronidase * T cell Subject RIV: FD - Oncology ; Hematology

  16. Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21 Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Sai Huang

    2016-01-01

    Conclusions: Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21 AML.

  17. Direct Human Papillomavirus E6 Whole-Cell Enzyme-Linked Immunosorbent Assay for Objective Measurement of E6 Oncoproteins in Cytology Samples

    OpenAIRE

    Yang, Yi-Shan; Smith-McCune, Karen; Darragh, Teresa M.; Lai, Yvonne; Lin, Ju-Hwa; Chang, Ting-Chang; Guo, Hsiao-Yun; Kesler, Tiea; Carter, Alicia; Castle, Philip E.; Cheng, Shuling

    2012-01-01

    A novel, whole-cell enzyme-linked immunosorbent assay (ELISA) based on a non-type-specific anti-human papillomavirus (HPV) E6 antibody was tested on 182 residual cytological specimens. For samples with a designation of more severe than cervical intraepithelial neoplasia grade 3 (CIN3+), 83% tested positive for E6; in a subset with paired testing for E6 ELISA and HPV DNA, 72% tested E6 positive and 92% tested high-risk (HR)-HPV DNA positive (P = 0.2). Among the women with a less than CIN3 diag...

  18. A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

    International Nuclear Information System (INIS)

    Wei, Na; Fan, Jun Kai; Gu, Jin Fa; He, Ling Feng; Tang, Wen Hao; Cao, Xin; Liu, Xin Yuan

    2009-01-01

    Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.

  19. Construction of an infectious clone of human adenovirus type 41.

    Science.gov (United States)

    Chen, Duo-Ling; Dong, Liu-Xin; Li, Meng; Guo, Xiao-Juan; Wang, Min; Liu, Xin-Feng; Lu, Zhuo-Zhuang; Hung, Tao

    2012-07-01

    Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

  20. Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung [Dong-A University College of Medicine, Pusan (Korea, Republic of)

    2003-02-01

    Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10{sup 11} particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy.

  1. Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

    International Nuclear Information System (INIS)

    Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung

    2003-01-01

    Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10 11 particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy

  2. Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control

    DEFF Research Database (Denmark)

    Sorensen, Maria R; Holst, Peter J; Pircher, Hanspeter

    2009-01-01

    of the vaccine antigen to invariant chain (Ii). To evaluate this strategy we used a mouse model, in which an immunodominant epitope (GP33) of the LCMV glycoprotein (GP) represents the tumor-associated neoantigen. Prophylactic vaccination of C57BL/6 mice with a replication-deficient human adenovirus 5 vector...... encoding GP linked to Ii (Ad-Ii-GP) resulted in complete protection against GP33-expressing B16.F10 tumors. Therapeutic vaccination with Ad-Ii-GP delayed tumor growth by more than 2 wk compared with sham vaccination. Notably, therapeutic vaccination with the linked vaccine was significantly better than...... the tumor degradation. Finally, Ad-Ii-GP but not Ad-GP vaccination can break the immunological non-reactivity in GP transgenic mice indicating that our vaccine strategy will prove efficient also against endogenous tumor antigens....

  3. Doxycycline-Regulated 3T3-L1 Preadipocyte Cell Line with Inducible, Stable Expression of Adenoviral E4orf1 Gene: A Cell Model to Study Insulin-Independent Glucose Disposal

    OpenAIRE

    Krishnapuram, Rashmi; Dhurandhar, Emily J.; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V.

    2013-01-01

    Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we establis...

  4. Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain

    DEFF Research Database (Denmark)

    Mikkelsen, Marianne; Holst, Peter Johannes; Bukh, Jens

    2011-01-01

    memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice...

  5. Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a “Bookmarking” Oncoprotein that Alters the Host Cell Epigenotype?

    Science.gov (United States)

    Niller, Hans Helmut; Minarovits, Janos

    2012-01-01

    EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may “bookmark” active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis. PMID:25436603

  6. The Major Histocompatibility Complex Class II Transactivator CIITA Inhibits the Persistent Activation of NF-κB by the Human T Cell Lymphotropic Virus Type 1 Tax-1 Oncoprotein.

    Science.gov (United States)

    Forlani, Greta; Abdallah, Rawan; Accolla, Roberto S; Tosi, Giovanna

    2016-01-20

    Human T cell lymphotropic virus type 1 (HTLV-1) Tax-1, a key protein in HTLV-1-induced T cell transformation, deregulates diverse cell signaling pathways. Among them, the NF-κB pathway is constitutively activated by Tax-1, which binds to NF-κB proteins and activates the IκB kinase (IKK). Upon phosphorylation-dependent IκB degradation, NF-κB migrates into the nucleus, mediating Tax-1-stimulated gene expression. We show that the transcriptional regulator of major histocompatibility complex class II genes CIITA (class II transactivator), endogenously or ectopically expressed in different cells, inhibits the activation of the canonical NF-κB pathway by Tax-1 and map the region that mediates this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained in the cytoplasm, and this correlates with impaired migration of RelA into the nucleus. Cytoplasmic and nuclear mutant forms of CIITA reveal that CIITA exploits different strategies to suppress Tax-1-mediated NF-κB activation in both subcellular compartments. CIITA interacts with Tax-1 without preventing Tax-1 binding to both IKKγ and RelA. Nevertheless, CIITA affects Tax-1-induced IKK activity, causing retention of the inactive p50/RelA/IκB complex in the cytoplasm. Nuclear CIITA associates with Tax-1/RelA in nuclear bodies, blocking Tax-1-dependent activation of NF-κB-responsive genes. Thus, CIITA inhibits cytoplasmic and nuclear steps of Tax-1-mediated NF-κB activation. These results, together with our previous finding that CIITA acts as a restriction factor inhibiting Tax-1-promoted HTLV-1 gene expression and replication, indicate that CIITA is a versatile molecule that might also counteract Tax-1 transforming activity. Unveiling the molecular basis of CIITA-mediated inhibition of Tax-1 functions may be important in defining new strategies to control HTLV-1 spreading and oncogenic potential. HTLV-1 is the causative agent of human adult T cell leukemia-lymphoma (ATLL). The viral transactivator Tax-1 plays a central role in the onset of ATLL, mostly by deregulating the NF-κB pathway. We demonstrate that CIITA, a key regulator of adaptive immunity, suppresses Tax-1-dependent activation of NF-κB by acting at several levels: it retains most of Tax-1 and RelA in the cytoplasm and inhibits their residual functional activity in the nucleus. Importantly, this inhibition occurs in cells that are targets of HTLV-1 infection. These findings are of interest in the field of virology because they expand the current knowledge of the functional relationship between viral products and cellular interactors and provide the basis for a better understanding of the molecular countermeasures adopted by the host cell to antagonize HTLV-1 spreading and transforming properties. Within this framework, our results may contribute to the establishment of novel strategies against HTLV-1 infection and virus-dependent oncogenic transformation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Human T-Cell Leukemia Virus Type I-Mediated Repression of PDZ-LIM Domain-Containing Protein 2 Involves DNA Methylation But Independent of the Viral Oncoprotein Tax

    Directory of Open Access Journals (Sweden)

    Pengrong Yan

    2009-10-01

    Full Text Available Human T-cell leukemia virus type I (HTLV-I is the etiological agent of adult T-cell leukemia (ATL. Our recent studies have shown that one important mechanism of HTLV-I-Mediated tumorigenesis is through PDZ-LIM domain-containing protein 2 (PDLIM2 repression, although the involved mechanism remains unknown. Here, we further report that HTLV-I-Mediated PDLIM2 repression was a pathophysiological event and the PDLIM2 repression involved DNA methylation. Whereas DNA methyltransferases 1 and 3b but not 3a were upregulated in HTLV-I-transformed T cells, the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC restored PDLIM2 expression and induced death of these malignant cells. Notably, the PDLIM2 repression was independent of the viral regulatory protein Tax because neither short-term induction nor long-term stable expression of Tax could downregulate PDLIM2 expression. These studies provide important insights into PDLIM2 regulation, HTLV-I leukemogenicity, long latency, and cancer health disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, these studies also suggest potential therapeutic strategies for ATL.

  8. Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

    Directory of Open Access Journals (Sweden)

    M.O. Diniz

    2011-05-01

    Full Text Available Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5 expressing three proteins (E7, E6, and E5 of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose induced a strong activation of E7-specific interferon-γ (INF-γ-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

  9. The PDZ domain binding motif (PBM) of human T-cell leukemia virus type 1 Tax can be substituted by heterologous PBMs from viral oncoproteins during T-cell transformation.

    Science.gov (United States)

    Aoyagi, Tomoya; Takahashi, Masahiko; Higuchi, Masaya; Oie, Masayasu; Tanaka, Yuetsu; Kiyono, Tohru; Aoyagi, Yutaka; Fujii, Masahiro

    2010-04-01

    Several tumor viruses, such as human T-cell leukemia virus (HTLV), human papilloma virus (HPV), human adenovirus, have high-oncogenic and low-oncogenic subtypes, and such subtype-specific oncogenesis is associated with the PDZ-domain binding motif (PBM) in their transforming proteins. HTLV-1, the causative agent of adult T-cell leukemia, encodes Tax1 with PBM as a transforming protein. The Tax1 PBM was substituted with those from other oncoviruses, and the transforming activity was examined. Tax1 mutants with PBM from either HPV-16 E6 or adenovirus type 9 E4ORF1 are fully active in the transformation of a mouse T-cell line from interleukin-2-dependent growth into independent growth. Interestingly, one such Tax1 PBM mutant had an extra amino acid insertion derived from E6 between PBM and the rest of Tax1, thus suggesting that the amino acid sequences of the peptides between PBM and the rest of Tax1 and the numbers only slightly affect the function of PBM in the transformation. Tax1 and Tax1 PBM mutants interacted with tumor suppressors Dlg1 and Scribble with PDZ-domains. Unlike E6, Tax1 PBM mutants as well as Tax1 did not or minimally induced the degradations of Dlg1 and Scribble, but instead induced their subcellular translocation from the detergent-soluble fraction into the insoluble fraction, thus suggesting that the inactivation mechanism of these tumor suppressor proteins is distinct. The present results suggest that PBMs of high-risk oncoviruses have a common function(s) required for these three tumor viruses to transform cells, which is likely associated with the subtype-specific oncogenesis of these tumor viruses.

  10. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Ran-yi; Zhou, Ling; Zhang, Yan-ling; Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min; Li, Li-xia; Fu, Xiang; Wu, Jiang-xue; Huang, Wenlin

    2013-01-01

    Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC

  11. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ran-yi, E-mail: liuranyi@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhou, Ling [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Zhang, Yan-ling [School of Biotechnology, Southern Medical University, Guangzhou 510515 (China); Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Li, Li-xia [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010 (China); Fu, Xiang; Wu, Jiang-xue [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Huang, Wenlin, E-mail: hwenl@mail.sysu.edu.cn [Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 510060 (China); Guangdong Provincial Key Laboratory of Tumor-Targeted Drug, Doublle Bioproducts Inc., Guangzhou 510663 (China)

    2013-12-13

    Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

  12. Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description

    Directory of Open Access Journals (Sweden)

    Rodrigo Pinheiro Araldi

    2015-01-01

    Full Text Available Bovine papillomavirus (BPV is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1 E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA and comet assay (CA. Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control. Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine.

  13. PPARγ2s A/B-domæne spiller en genspecifik rolle i transaktivering og co-faktor rekruttering

    DEFF Research Database (Denmark)

    Bugge, Anne Skovsø; Grøntved, Lars; M. Aagaard, Mads

    We have previously shown that adenoviral expression of PPARs leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5-8 hours following transduction (Nielsen at al. 2006). Here we have used the adenoviral delivery system to identify nove...... project X-TRA-NET....

  14. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  15. Imaging analysis of nuclear antiviral factors through direct detection of incoming adenovirus genome complexes

    Energy Technology Data Exchange (ETDEWEB)

    Komatsu, Tetsuro [Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, Université de Bordeaux, Bordeaux 33076 (France); Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575 (Japan); Will, Hans [Department of Tumor Biology, University Hospital Hamburg-Eppendorf, 20246 Hamburg (Germany); Nagata, Kyosuke [Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575 (Japan); Wodrich, Harald, E-mail: harald.wodrich@u-bordeaux.fr [Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, Université de Bordeaux, Bordeaux 33076 (France)

    2016-04-22

    Recent studies involving several viral systems have highlighted the importance of cellular intrinsic defense mechanisms through nuclear antiviral proteins that restrict viral propagation. These factors include among others components of PML nuclear bodies, the nuclear DNA sensor IFI16, and a potential restriction factor PHF13/SPOC1. For several nuclear replicating DNA viruses, it was shown that these factors sense and target viral genomes immediately upon nuclear import. In contrast to the anticipated view, we recently found that incoming adenoviral genomes are not targeted by PML nuclear bodies. Here we further explored cellular responses against adenoviral infection by focusing on specific conditions as well as additional nuclear antiviral factors. In line with our previous findings, we show that neither interferon treatment nor the use of specific isoforms of PML nuclear body components results in co-localization between incoming adenoviral genomes and the subnuclear domains. Furthermore, our imaging analyses indicated that neither IFI16 nor PHF13/SPOC1 are likely to target incoming adenoviral genomes. Thus our findings suggest that incoming adenoviral genomes may be able to escape from a large repertoire of nuclear antiviral mechanisms, providing a rationale for the efficient initiation of lytic replication cycle. - Highlights: • Host nuclear antiviral factors were analyzed upon adenovirus genome delivery. • Interferon treatments fail to permit PML nuclear bodies to target adenoviral genomes. • Neither Sp100A nor B targets adenoviral genomes despite potentially opposite roles. • The nuclear DNA sensor IFI16 does not target incoming adenoviral genomes. • PHF13/SPOC1 targets neither incoming adenoviral genomes nor genome-bound protein VII.

  16. Imaging analysis of nuclear antiviral factors through direct detection of incoming adenovirus genome complexes

    International Nuclear Information System (INIS)

    Komatsu, Tetsuro; Will, Hans; Nagata, Kyosuke; Wodrich, Harald

    2016-01-01

    Recent studies involving several viral systems have highlighted the importance of cellular intrinsic defense mechanisms through nuclear antiviral proteins that restrict viral propagation. These factors include among others components of PML nuclear bodies, the nuclear DNA sensor IFI16, and a potential restriction factor PHF13/SPOC1. For several nuclear replicating DNA viruses, it was shown that these factors sense and target viral genomes immediately upon nuclear import. In contrast to the anticipated view, we recently found that incoming adenoviral genomes are not targeted by PML nuclear bodies. Here we further explored cellular responses against adenoviral infection by focusing on specific conditions as well as additional nuclear antiviral factors. In line with our previous findings, we show that neither interferon treatment nor the use of specific isoforms of PML nuclear body components results in co-localization between incoming adenoviral genomes and the subnuclear domains. Furthermore, our imaging analyses indicated that neither IFI16 nor PHF13/SPOC1 are likely to target incoming adenoviral genomes. Thus our findings suggest that incoming adenoviral genomes may be able to escape from a large repertoire of nuclear antiviral mechanisms, providing a rationale for the efficient initiation of lytic replication cycle. - Highlights: • Host nuclear antiviral factors were analyzed upon adenovirus genome delivery. • Interferon treatments fail to permit PML nuclear bodies to target adenoviral genomes. • Neither Sp100A nor B targets adenoviral genomes despite potentially opposite roles. • The nuclear DNA sensor IFI16 does not target incoming adenoviral genomes. • PHF13/SPOC1 targets neither incoming adenoviral genomes nor genome-bound protein VII.

  17. Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV Construção de vetores adenovirais expressando as glicoproteínas F e G de vírus respiratório sincicial humano (HRSV

    Directory of Open Access Journals (Sweden)

    Ithana Monteiro Kosaka

    2004-06-01

    Full Text Available Human Respiratory Syncytial Virus (HRSV was first characterized in 1957 and has since been recognized as the most common viral cause of severe respiratory tract infection in young infants worldwide. Despite many years of research there is still no effective treatment or any immediate prospect of a vaccine. The HRSV genome is composed of single stranded negative sense RNA and the virion consists of a nucleocapsid packaged within a lipid envelope. The envelope contains spike-like projections, each being a homo-oligomer of one of three transmembrane viral envelope proteins: the attachment protein G, the fusion protein F involved in viral penetration and the small hydrofobic protein SH. The aim of this work was to construct two recombinant replication-defective adenoviruses carrying separately F and G genes from HRSV. This system was chosen because adenovirus delivers genes into target cells with high efficiency in a variety of cell lines and can be used in vitro and in vivo. In order to obtain the recombinant viruses, we did RT-PCR of RNA extracted from the HRSV A2 strain, the genes F and G were cloned in to pAdeno-X vectors. pAdeno-F and pAdeno-G were transfected in HEK-293 cells for the production of recombinant viruses, that expressed efficiently these two proteins and provide us the means for doing functional assays and immunization tests.O Vírus Sincicial Respiratório Humano (HRSV foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment", a proteína de fusão F ("fusion" e a proteína SH ("small hydrofobic". O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.

  18. Recombinant fibromodulin has therapeutic effects on diabetic nephropathy by down-regulating transforming growth factor-β1 in streptozotocin-induced diabetic rat model

    Directory of Open Access Journals (Sweden)

    Maryam Foroutan Jazi

    2016-03-01

    Conclusion:Intraperitoneal injection of adenoviral vectors expressing fibromodulin  reduced TGF-β1 level in diabetic rat models. The molecular mechanisms involved in this process require further study.

  19. Promoting effect of adipocytokine, apelin, on hepatic injury in ...

    African Journals Online (AJOL)

    Marwa N. Emam

    2015-12-14

    Dec 14, 2015 ... and inflammatory markers, pancreatic and hepatic MPO activity with ..... ment, and possibly mediated by an apelin-induced reduction ... So targeting the ape- .... Adenoviral delivery of human and viral IL-10 in murine sepsis.

  20. S14 as a Therapeutic Target in Breast Cancer

    National Research Council Canada - National Science Library

    Kinlaw, William

    2004-01-01

    .... Our aims are first to develop a model of anti-S14 breast cancer therapy in mice. Intratumoral adenoviral delivery of an S14-antisense gene into human breast cancer cell xenografts caused a significant inhibition of tumor growth...

  1. The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

    Directory of Open Access Journals (Sweden)

    Pierdomenico Ruggeri

    Full Text Available The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs, correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

  2. Lymphatic Vascular-Based Therapy for IBD

    Science.gov (United States)

    2012-07-01

    significant preservation of crypts and epithelium. Adenoviral VEGF-C + DSS also significantly reduced histopathological injury produced DSS but to a... preserved than in VEGF-D. Adenoviral GFP + DSS did not provide significant protection against tissue injury (29 ± 2.3, not significant different vs. DSS...male or female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) or kB- lacZ mice (provided by Dr. Sylvie Memet, Pasteur Institute, Paris, France

  3. Inhibitory effect of PTD-OD-HA fusion protein on Bcr-Abl in K562 cells

    Directory of Open Access Journals (Sweden)

    Miao GAO

    2012-10-01

    Full Text Available Objective To study the transduction dynamics, location of PTD-OD-HA fusion protein and its interaction with Bcr-Abl oncoprotein in K562 cell lines, and explore the influence of PTD-OD-HA fusion protein on oligomerization and tyrosine kinase activity of Bcr-Abl. Methods PTD-OD-HA fusion protein was labeled with FITC and co-cultured with K562 cells. The transduction efficiency of labeled PTD-OD-HA at different doses and time intervals was observed under fluorescence microscope. The location of labeled PTD-OD-HA fusion protein in K562 cells was detected by confocal microscopy. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was confirmed by coimmunoprecipitation. The phosphorylation of Bcr-Abl oncoprotein was detected by Western blotting. Results PTD-OD-HA fusion protein labeled with FITC was transduced into K562 cells in a dose- and time-dependent manner. PTD-OD-HA fusion protein was located in the cytoplasm of K562 cells and was consistent with the location of Bcr-Abl oncoprotein. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was proved in K562 cells. This interaction could interrupt the homologous oligomerization of Bcr-Abl oncoprotein and reduce the phosphorylation of Bcr-Abl oncoprotein. Conclusion PTD-OD-HA fusion protein could be transduced into K562 cells efficiently, inhibit the oligomerization and reduce the phosphorylation of Bcr-Abl oncoprotein.

  4. Gene therapy in the inner ear using adenovirus vectors.

    Science.gov (United States)

    Husseman, Jacob; Raphael, Yehoash

    2009-01-01

    Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression. Copyright (c) 2009 S. Karger AG, Basel.

  5. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes

    2012-01-01

    directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated...... that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD......8 T-cell memory even in individuals with pre-existing vector immunity....

  6. Future prospects for the development of cost-effective Adenovirus vaccines

    DEFF Research Database (Denmark)

    Fougeroux, Cyrielle; Holst, Peter J

    2017-01-01

    -vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient...... as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes...

  7. An Imaging System to Monitor Efficacy of Adenovirus-Based Virotherapy Agents

    Science.gov (United States)

    2006-02-01

    Izumo, S. (2000). Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy . Nat Med 6(5), 482-483. 20 Kelkar, S. A...for adenoviral transduction of canine osteosarcoma cells. Gene Ther. 2006 Mar;13(5):389-99. 3. Le LP, Le HN, N elson AM, Yamamoto M, Curiel DT...Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells LP Le1,4, AA Rivera1,4, JN Glasgow1, VV Ternovoi1, H Wu1, M Wang1, BF

  8. Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

    Science.gov (United States)

    Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

    2017-12-07

    The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

  9. v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII

    DEFF Research Database (Denmark)

    Disela, C; Glineur, C; Bugge, T

    1991-01-01

    The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte...... of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein....

  10. Effect of Skeletal Muscle Na+ Channel Delivered Via a Cell Platform on Cardiac Conduction and Arrhythmia Induction

    NARCIS (Netherlands)

    Boink, Gerard J. J.; Lu, Jia; Driessen, Helen E.; Duan, Lian; Sosunov, Eugene A.; Anyukhovsky, Evgeny P.; Shlapakova, Iryna N.; Lau, David H.; Rosen, Tove S.; Danilo, Peter; Jia, Zhiheng; Ozgen, Nazira; Bobkov, Yevgeniy; Guo, Yuanjian; Brink, Peter R.; Kryukova, Yelena; Robinson, Richard B.; Entcheva, Emilia; Cohen, Ira S.; Rosen, Michael R.

    2012-01-01

    Background-In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel is largely inactivated, contributing to slow conduction and reentry. We have demonstrated that adenoviral delivery of the skeletal muscle Na+ channel (SkM1) to epicardial border zones normalizes

  11. characterisation of gastro- enteritis-associated adenoviruses in ...

    African Journals Online (AJOL)

    Objective. To analyse adenovirus (Ad) numbers and types associated with paediatric gastro-enteritis in South Africa. Setting. Gauteng, 1994-1996. Metfwds. A total of 234 paediatric diarrhoeal stool samples were screened for Ad using commercial enzyme-linked. iInmunosorbent assays (EUSAs). Adenoviral isolates were.

  12. Detection of adenovirus hexon sequence in a cat by polymerase chain reaction(short communication)

    NARCIS (Netherlands)

    Horzinek, M.C.; Lakatos, B.; Farkas, J.; Egberink, H.F.; Vennema, H.; Benko, M.

    1999-01-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal

  13. PB1 as a potential target for increasing the breadth of T-cell mediated immunity to Influenza A

    DEFF Research Database (Denmark)

    Uddbäck, Ida E M; Steffensen, Maria A; Pedersen, Sara R

    2016-01-01

    Recently, we showed that combined intranasal and subcutaneous immunization with a non-replicating adenoviral vector expressing NP of influenza A, strain PR8, induced long-standing protection against a range of influenza A viruses. However, H-2(b) mice challenged with an influenza A strain mutated...

  14. Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats.

    Science.gov (United States)

    Huang, Shun-Ping; Lin, Po-Kang; Liu, Jorn-Hon; Khor, Chin-Ni; Lee, Yih-Jing

    2004-01-01

    Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats. Copyright 2004 National Science Council, ROC and S. Karger AG, Basel

  15. Effects of infection with recombinant adenovirus on human vascular endothelial and smooth muscle cells

    NARCIS (Netherlands)

    Quax, P.H.A.; Lamfers, M.L.M.; Grimbergen, J.M.; Teeling, J.; Hoeben, R.C.; Nieuw Amerongen, G.P. van; Hinsbergh, V.W.M. van

    1996-01-01

    The plasminogen activation (PA) system is involved in vascular remodelling. Modulating its activity in vascular cells might be a way to interfere in processes such as angiogenesis and restenosis. Adenoviral vectors have become a favourable tool for direct gene transfer into vascular cells. In the

  16. Screening for adenoviruses in haematological neoplasia: High prevalence in mantle cell lymphoma.

    Science.gov (United States)

    Kosulin, Karin; Rauch, Margit; Ambros, Peter F; Pötschger, Ulrike; Chott, Andreas; Jäger, Ulrich; Drach, Johannes; Nader, Alexander; Lion, Thomas

    2014-02-01

    Human adenoviruses possess oncogenic capacity which is well documented in mammalian animal models, but their possible implication in human malignancy has remained enigmatic. Following primary infection, adenoviruses can persist in a latent state in lymphocytes where the virus is apparently able to evade immune surveillance. In the present study, we have employed a broad-spectrum adenovirus polymerase chain reaction (PCR) assay to systematically screen more than 200 diagnostic specimens of different lymphoid malignancies including acute lymphocytic leukaemia (n=50), chronic lymphocytic leukaemia (n=50), various types of malignant lymphoma (n=100) and multiple myeloma (n=11) for the presence of adenoviral sequences. While most entities analysed revealed negative findings in virtually all specimens tested, adenoviral DNA was detected in 15/36 (42%) mantle cell lymphomas investigated. The most prevalent adenoviral species detected was C, and less commonly B. Adenovirus-positive findings in patients with mantle cell lymphoma were made at different sites including bone marrow (n=7), intestine (n=5), lymph nodes (n=2) and tonsillar tissue (n=1). The presence of adenoviral sequences identified by PCR was confirmed in individual cells by fluorescence in-situ hybridisation (FISH). The frequent observation of adenoviruses in mantle cell lymphoma is intriguings, and raises questions about their possible involvement in the pathogenesis of this lymphoid malignancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Fatal adenovirus encephalomyeloradiculitis in an umbilical cord stem cell transplant recipient

    OpenAIRE

    Awosika, Oluwole O.; Lyons, Jennifer L.; Ciarlini, Pedro; Phillips, Richard E.; Alfson, Elizabeth D.; Johnson, Emily L.; Koo, Sophia; Marty, Francisco; Drew, Clifton; Zaki, Sherif; Folkerth, Rebecca D.; Klein, Joshua P.

    2013-01-01

    Adenovirus infections frequently complicate allogeneic stem cell transplants but nervous system involvement, usually presenting as encephalitis, is atypical. Progression from encephalitis to myeloradiculitis has not been described previously.1 We present a unique case of fatal adenoviral encephalomyeloradiculitis with imaging and pathologic correlates.

  18. Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma

    NARCIS (Netherlands)

    Kuhlmann, K.F.D.; Geer, M.A. van; Bakker, C.T.; Dekker, J.E.M.; Havenga, M.J.E.; Oude Elferink, R.P.J.; Gouma, D.J.; Bosma, P.J.; Wesseling, J.G.

    2009-01-01

    Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved

  19. [Use of polymeric suspensions as a viral sorbent to detect cattle serum antibodies].

    Science.gov (United States)

    Stanishevskiĭ, Ia M; Lobova, T P; Gritskova, I A; Belousova, R V; Prokopov, N I; Tret'iakova, I V; Tkalia, E E

    2006-01-01

    The paper shows it possible to use stained polymeric microspheres, 1.7 microm in diameter, that contain viruses onto the surface, in the latex agglutination test to detect antibodies to the bovine serum viruses of infective rhinotracheitis, parainfluenza-3, viral diarrhea, respiratory syncytial infection, and adenoviral infection.

  20. MHC class II-associated invariant chain linkage of antigen dramatically improves cell-mediated immunity induced by adenovirus vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Mandrup Jensen, Camilla Maria; Orskov, Cathrine

    2008-01-01

    The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most...

  1. Gene delivery to pancreatic exocrine cells in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Houbracken Isabelle

    2012-10-01

    Full Text Available Abstract Background Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. Results For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. Conclusions In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.

  2. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

    Directory of Open Access Journals (Sweden)

    Maria Abildgaard Steffensen

    Full Text Available Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii. To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

  3. http://www.bioline.org.br/js 37 The Prevalence Overexpression Of C ...

    African Journals Online (AJOL)

    jen

    The Prevalence Overexpression Of C-Erbb-2 Oncoprotein In Carcinoma Of The Prostate-. Mulago Hospital. R. Alenyo1, M. Odida2, S Watya1. 1Department of Surgery, 2Department of Pathology, Makerere University, Kampala – Uganda. Correpondence to: Dr. Rose Alenyo, Email: Rose Alenyo ,.

  4. Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells

    DEFF Research Database (Denmark)

    Hjerrild, M; Milne, D; Dumaz, N

    2001-01-01

    Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteasome. Stress signals which impinge on p53, leading to its...

  5. The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs

    NARCIS (Netherlands)

    Mandoli, Amit; Singh, Abhishek A.; Prange, Koen H. M.; Tijchon, Esther; Oerlemans, Marjolein; Dirks, Rene; Ter Huurne, Menno; Wierenga, Albertus T. J.; Janssen-Megens, Eva M.; Berentsen, Kim; Sharifi, Nilofar; Kim, Bowon; Matarese, Filomena; Nguyen, Luan N.; Hubner, Nina C.; Rao, Nagesha A.; van den Akker, Emile; Altucci, Lucia; Vellenga, Edo; Stunnenberg, Hendrik G.; Martens, Joost H. A.

    2016-01-01

    The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels

  6. Chromatin-bound MDM2, a new player in metabolism.

    Science.gov (United States)

    Riscal, Romain; Le Cam, Laurent; Linares, Laetitia K

    2016-01-01

    The oncoprotein MDM2 is recognized as a major negative regulator of the p53 tumor suppressor but growing evidence indicates that its oncogenic activities extend beyond p53. We show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis.

  7. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-04

    Feb 4, 2009 ... targeting the viral oncoproteins E6 and E7 are markers for HPV-associated ... Luminex XYP plate handler, Luminex SD sheath fluid delivery system, a Pentium 4 .... expression mediated by a potato virus X derived vector of the E7 protein .... inflammation, and antioxidant nutrients – assessing their roles as.

  8. Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation

    NARCIS (Netherlands)

    Rahimian, Sima; Fransen, Marieke F.; Kleinovink, Jan Willem; Christensen, Jonatan Riis; Amidi, Maryam|info:eu-repo/dai/nl/304834912; Hennink, Wim E.|info:eu-repo/dai/nl/070880409; Ossendorp, Ferry

    2015-01-01

    The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In

  9. East African Medical Journal - Vol 83, No 8 (2006)

    African Journals Online (AJOL)

    Oncoprotein over-expression in breast cancer and its relationship to histology and grade in a Ugandan population · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. H Nalwoga, M Odida, H Wabinga, 411-415. http://dx.doi.org/10.4314/eamj.v83i8.9454 ...

  10. The retinoblastoma protein as a transcriptional repressor

    DEFF Research Database (Denmark)

    Helin, K; Ed, H

    1993-01-01

    The retinoblastoma protein (pRB) is one of the best-studied tumour suppressor gene products. Its loss during the genesis of many human tumours, its inactivation by several DNA tumour virus oncoproteins, and its ability to inhibit cell growth when introduced into dividing cells all suggest that p...

  11. Evaluation of immunological cross-reactivity between clade A9 high-risk human papillomavirus types on the basis of E6-Specific CD4+ memory T cell responses

    NARCIS (Netherlands)

    van den Hende, Muriel; Redeker, Anke; Kwappenberg, Kitty M. C.; Franken, Kees L. M. C.; Drijfhout, Jan W.; Oostendorp, Jaap; Valentijn, A. Rob P. M.; Fathers, Loraine M.; Welters, Marij J. P.; Melief, Cornelis J. M.; Kenter, Gemma G.; van der Burg, Sjoerd H.; Offringa, Rienk

    2010-01-01

    CD4(+) T cell responses against the E6 oncoprotein of human papillomavirus (HPV) type 16 and 5 closely related members of clade A9 (HPV31, 33, 35, 52, and 58) were charted in peripheral blood mononuclear cell cultures from healthy subjects and patients who underwent HPV16 E6/E7-specific vaccination.

  12. Human Papilloma Virus 16 E6 RNA Interference Enhances Cisplatin and Death Receptor-Mediated Apoptosis in Human Cervical Carcinoma Cells

    NARCIS (Netherlands)

    Tan, Shinta; Hougardy, Brigitte M. T.; Meersma, Gert J.; Schaap, Bessel; de Vries, Elisabeth G. E.; van der Zee, Ate G. J.; de Jong, Steven

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small

  13. Superior therapeutic efficacy of alphavirus-mediated immunization against human papilloma virus type 16 antigens in a murine tumour model : effects of the route of immunization

    NARCIS (Netherlands)

    Daemen, T; Riezebos-Brilman, A; Regts, J; Dontje, B; van der Zee, A; Wilschut, J

    2004-01-01

    In our efforts to develop a strong, effective immune response against cervical carcinoma and premalignant disease, we study the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPVs). Optimal immunization conditions are

  14. Rapid enrichment of human papillomavirus (HPV)-specific polyclonal T cell populations for adoptive immunotherapy of cervical cancer

    NARCIS (Netherlands)

    de Jong, Annemieke; van der Hulst, Jeanette M.; Kenter, Gemma G.; Drijfhout, Jan Wouter; Franken, Kees L. M. C.; Vermeij, Pieter; Offringa, Rienk; van der Burg, Sjoerd H.; Melief, Cornelis J. M.

    2005-01-01

    The majority of cervical cancers are caused by human papillomavirus type 16 (HPV16). Cervical cancer is associated with an ineffective host immune response against the HPV16 oncoproteins, characterized by the lack of the strong E6-specific T-helper type 1 (Th1) immunity that is generally present in

  15. Effect of His(6)-tagging of anterior gradient 2 protein on its electro-oxidation

    Czech Academy of Sciences Publication Activity Database

    Ostatná, Veronika; Vargová, Veronika; Hrstka, R.; Durech, M.; Vojtešek, B.; Paleček, Emil

    2014-01-01

    Roč. 150, DEC2014 (2014), s. 218-222 ISSN 0013-4686 R&D Projects: GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : Anterior Gradient 2?oncoprotein * His-tagged proteins * carbon electrodes Subject RIV: BO - Biophysics Impact factor: 4.504, year: 2014

  16. Survivin is a therapeutic target in Merkel cell carcinoma

    NARCIS (Netherlands)

    Arora, Reety; Shuda, Masahiro; Guastafierro, Anna; Feng, Huichen; Toptan, Tuna; Tolstov, Yanis; Normolle, Daniel; Vollmer, Laura L; Vogt, Andreas; Dömling, Alexander; Brodsky, Jeffrey L; Chang, Yuan; Moore, Patrick S

    2012-01-01

    Merkel cell polyomavirus (MCV) causes ~80% of primary and metastatic Merkel cell carcinomas (MCCs). By comparing digital transcriptome subtraction deep-sequencing profiles, we found that transcripts of the cellular survivin oncoprotein [BIRC5a (baculoviral inhibitor of apoptosis repeat-containing

  17. Analysis of the ARF/p53 Pathway During Oncogenic Stimulation

    National Research Council Canada - National Science Library

    Nahle, Zaher

    2003-01-01

    ... or deficient for the ARF and/or p53 genes. We found that the ElA oncoprotein regulates the expression of a myriad of targets involved in a diversity of functions such as apoptosis, cell cycle progression, checkpoint control, DNA replication...

  18. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

    Science.gov (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

    2016-02-01

    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  19. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  20. Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts

    Directory of Open Access Journals (Sweden)

    Jing Li Huang

    2016-09-01

    Full Text Available Oncolytic adenoviruses (OAds are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor, interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

  1. Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts.

    Science.gov (United States)

    Huang, Jing Li; LaRocca, Christopher J; Yamamoto, Masato

    2016-09-19

    Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

  2. Early life vaccination

    DEFF Research Database (Denmark)

    Nazerai, Loulieta; Bassi, Maria Rosaria; Uddbäck, Ida Elin Maria

    2016-01-01

    Intracellular pathogens represent a serious threat during early life. Importantly, even though the immune system of newborns may be characterized as developmentally immature, with a propensity to develop Th2 immunity, significant CD8+ T-cell responses may still be elicited in the context of optimal...... the first period of life and provide a pertinent alternative in infant vaccinology. To address this, infant mice were vaccinated with three different adenoviral vectors and the CD8+ T-cell response after early life vaccination was explored. We assessed the frequency, polyfunctionality and in vivo...... cytotoxicity of the elicited memory CD8+ T cells, as well as the potential of these cells to respond to secondary infections and confer protection. We further tested the impact of maternal immunity against our replication-deficient adenoviral vector during early life vaccination. Overall, our results indicate...

  3. Targeting of non-dominant antigens as a vaccine strategy to broaden T-cell responses during chronic viral infection

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Jensen, Benjamin Anderschou Holbech; Ragonnaud, Emeline

    2015-01-01

    In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilita......In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes...... was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted...

  4. Systemic adenovirus infection in Bearded Dragons (Pogona vitticeps): histological, ultrastructural and molecular findings.

    Science.gov (United States)

    Moormann, S; Seehusen, F; Reckling, D; Kilwinski, J; Puff, C; Elhensheri, M; Wohlsein, P; Peters, M

    2009-07-01

    Three Inland Bearded Dragons (Pogona vitticeps) from two breeding groups were humanely destroyed following a period of anorexia. Two of the animals were 8-months old and related and one animal was approximately 2-weeks old. Necropsy examination revealed poor bodily condition but no other gross abnormalities. Microscopically there was non-suppurative hepatitis and interstitial nephritis. Multiple large, amphophilic, intranuclear inclusion bodies were present within hepatocytes and epithelial cells of the bile ducts, renal tubules, small and large intestinal mucosa, pancreatic acini and oral mucous membranes. Transmission electron microscopy (TEM) demonstrated that the inclusions comprised viral particles with morphology consistent with an adenovirus. A fragment of the adenoviral polymerase gene was amplified, sequenced and compared with other reptilian adenoviral sequences.

  5. Necrotizing hepatitis in a domestic pigeon (Columba livia).

    Science.gov (United States)

    Himmel, L; O'Connor, M; Premanandan, C

    2014-11-01

    An adult male domestic pigeon (Columba livia) was presented for necropsy following natural death after a period of chronic weight loss and severe intestinal ascariasis. Histopathologic examination of the liver found moderate to marked, multifocal necrotizing hepatitis with large, basophilic intranuclear inclusion bodies. Transmission electron microscopy of affected hepatocytes demonstrated numerous intra- and perinuclear icosahedral virions arranged in a lattice structure, consistent with adenoviral infection. © The Author(s) 2014.

  6. RKIP phosphorylation–dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3

    Energy Technology Data Exchange (ETDEWEB)

    Hahm, Jong Ryeal [Department of Internal Medicine, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Ahmed, Mahmoud [Department of Biochemistry and Convergence Medical Science, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr [Department of Biochemistry and Convergence Medical Science, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of); Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27 (Korea, Republic of)

    2016-09-09

    3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-L-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 was highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-L-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity. - Highlights: • Overexpression of GFP-LC3 in 3T3-L1 cells produces intracellular lipid droplets. • SREBP2 is highly activated in preadipocytes transfected with adenoviral GFP-LC3. • RKIP phosphorylation at serine 153 is significantly increased during adipogenesis. • RKIP knockdown promotes ERK1 and PPARγ activation during adipogenesis. • RKIP-dependent ERK1 activation increases triacylglycerides in

  7. Central melanin-concentrating hormone influences liver and adipose metabolism via specific hypothalamic nuclei and efferent autonomic/JNK1 pathways.

    OpenAIRE

    Imbernon, Monica; Beiroa, Daniel; Vázquez, María J.; Morgan, Donald A.; Veyrat–Durebex, Christelle; Porteiro, Begoña; Díaz–Arteaga, Adenis; Senra, Ana; Busquets, Silvia; Velásquez, Douglas A.; Al–Massadi, Omar; Varela, Luis; Gándara, Marina; López–Soriano, Francisco–Javier; Gallego, Rosalía

    2013-01-01

    BACKGROUND AIMS Specific neuronal circuits modulate autonomic outflow to liver and white adipose tissue. Melanin concentrating hormone (MCH) deficient mice are hypophagic lean and do not develop hepatosteatosis when fed a high fat diet. Herein we sought to investigate the role of MCH an orexigenic neuropeptide specifically expressed in the lateral hypothalamic area on hepatic and adipocyte metabolism. METHODS Chronic central administration of MCH and adenoviral vectors increasing MCH sign...

  8. Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

    Science.gov (United States)

    Jing, Yanyan; Liu, Wei; Cao, Hongchao; Zhang, Duo; Yao, Xuan; Zhang, Shengjie; Xia, Hongfeng; Li, Dan; Wang, Yu-cheng; Yan, Jun; Hui, Lijian; Ying, Hao

    2015-06-01

    It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor β-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  9. The Modulation of Fibrosis in Scleroderma by 3-Deoxyglucosone

    Science.gov (United States)

    2010-06-01

    nonenzymatic Maillard reaction between proteins and re- active carbonyl and dicarbonyl compounds.3,4 Degrada- tion of glycated proteins subsequently results...ER stress-induced apoptosis marker GADD153 is upregulated in fibroblasts cultured on 3DG- collagen Accumulation of misfolded proteins within the ER...Yu FS, Castro MG, Ljubimov AV. Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery

  10. Respiratory syncytial virus, adenoviruses, and mixed acute lower respiratory infections in children in a developing country.

    Science.gov (United States)

    Rodríguez-Martínez, Carlos E; Rodríguez, Diego Andrés; Nino, Gustavo

    2015-05-01

    There is growing evidence suggesting greater severity and worse outcomes in children with mixed as compared to single respiratory virus infections. However, studies that assess the risk factors that may predispose a child to a mixture of respiratory syncytial virus (RSV) and adenoviral infections, are scarce. In a retrospective cohort study, the study investigated the epidemiology of RSV and adenovirus infections and predictors of mixed RSV-adenoviral infections in young children hospitalized with acute lower respiratory infection in Bogota, Colombia, South America, over a 2-year period 2009-2011. Of a total of 5,539 children admitted with a diagnosis of acute lower respiratory infection, 2,267 (40.9%) who were positive for RSV and/or adenovirus were selected. Out the total number of cases, 1,416 (62.5%) infections occurred during the 3-month period from March to May, the first rainy season of Bogota, Colombia. After controlling for gender, month when the nasopharyngeal sample was taken, and other pre-existing conditions, it was found that an age greater than 6 months (OR:1.74; CI 95%:1.05-2.89; P = 0.030) and malnutrition as a comorbidity (OR:9.92; CI 95%:1.01-100.9; P = 0.049) were independent predictors of mixed RSV-adenoviral infections in the sample of patients. In conclusion, RSV and adenovirus are significant causes of acute lower respiratory infection in infants and young children in Bogota, Colombia, especially during the first rainy season. The identified predictors of mixed RSV-adenoviral infections should be taken into account when planning intervention, in order to reduce the burden of acute lower respiratory infection in young children living in the country. © 2015 Wiley Periodicals, Inc.

  11. Efectos morfológicos de la terapia génica restauradora de la timulina sobre la adenohipófisis de ratones atímicos

    OpenAIRE

    Martines, Eliana

    2012-01-01

    El objetivo general de la presente tesis es implementar una terapia génica pituitaria neonatal mediante el vector adenoviral portador del gen sintético para timulina (RAd- FTS), con el fin de prevenir deficiencias en las poblaciones adenohipofisarias, en los niveles séricos de la timulina y de las hormonas hipofisarias de ratones nude Facultad de Ciencias Médicas

  12. Vestibular regeneration ? experimental models and clinical implications

    OpenAIRE

    Albu, Silviu; Muresanu, Dafin F

    2012-01-01

    Therapies aimed at the protection and/or regeneration of inner ear hair cells are of great interest, given the significant monetary and quality of life impact of balance disorders. Different viral vectors have been shown to transfect various cell types in the inner ear. The past decade has provided tremendous advances in the use of adenoviral vectors to achieve targeted treatment delivery. Several routes of delivery have been identified to introduce vectors into the inner ear while minimizing...

  13. Comparison of Vaccine-Induced Effector CD8 T Cell Responses Directed against Self- and Non-Self-Tumor Antigens

    DEFF Research Database (Denmark)

    Pedersen, Sara R; Sørensen, Maria R; Buus, Søren

    2013-01-01

    It is generally accepted that CD8 T cells play a major role in tumor control, yet vaccination aimed at eliciting potent CD8 T cell responses are rarely efficient in clinical trials. To try and understand why this is so, we have generated potent adenoviral vectors encoding the endogenous tumor Ags...... that low avidity of the self-TA-specific CD8 T cells may represent a major obstacle for efficient immunotherapy of cancer....

  14. Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

    Science.gov (United States)

    Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

    2017-12-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

  15. Hemorrhagic and necrotizing hepatitis associated with administration of a modified live canine adenovirus-2 vaccine in a maned wolf (Chrysocyon brachyurus).

    Science.gov (United States)

    Swenson, Julie; Orr, Kathryn; Bradley, Gregory A

    2012-06-01

    A 15-yr-old, female, maned wolf (Chrysocyon brachyurus) was euthanized after presenting semicomatose with severe, uncontrolled frank hemorrhage from her rectum 6 days following a routine physical examination and vaccination. Histopathology indicated severe hemorrhagic and necrotizing hepatitis with intranuclear basophilic inclusion bodies in the liver that were thought to be consistent with adenoviral infection. Further classification by polymerase chain reaction, immunohistochemical staining, virus isolation, and electron microscopy confirmed the etiologic agent to be canine adenovirus-2. A representative sample of the vaccine that had been used was submitted and sequenced along with the virus isolated from the maned wolf. The sequencing of the etiologic agent that had been isolated from the maned wolf was determined to be the same as the strain of virus used in the production of the modified live vaccine that had been administered 6 days prior to death. From this information, the diagnosis of vaccine-induced adenoviral hepatitis was made. This is the first confirmed case of vaccine-induced canine adenoviral hepatitis in a maned wolf.

  16. [Construction and expression of a recombinant adenovirus with LZP3].

    Science.gov (United States)

    Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

    2007-08-01

    To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

  17. Vestibular regeneration--experimental models and clinical implications.

    Science.gov (United States)

    Albu, Silviu; Muresanu, Dafin F

    2012-09-01

    Therapies aimed at the protection and/or regeneration of inner ear hair cells are of great interest, given the significant monetary and quality of life impact of balance disorders. Different viral vectors have been shown to transfect various cell types in the inner ear. The past decade has provided tremendous advances in the use of adenoviral vectors to achieve targeted treatment delivery. Several routes of delivery have been identified to introduce vectors into the inner ear while minimizing injury to surrounding structures. Recently, the transcription factor Atoh1 was determined to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo has demonstrated the ability to regenerate vestibular hair cells by causing transdifferentiation of neighbouring epithelial-supporting cells. Functional recovery of the vestibular system has also been documented following adenoviral-induced Atoh1 overexpression. Experiments demonstrating gene transfer in human vestibular epithelial cells reveal that the human inner ear is a suitable target for gene therapy. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  18. [Hemorrhagic cystitis due to adenovirus in a renal transplant recipient: the first reported case in black Africa in a setting of a very beginning of a kidney transplantation program and review of the literature].

    Science.gov (United States)

    Ackoundou-N'Guessan, Clément; Coulibaly, Noël; Guei, Cyr Monley; Aye, Denis; N'guessan, Francis Yapi; N'Dah, Justin Kouame; Lagou, Delphine Amélie; Tia, Mélanie Weu; Coulibaly, Pessa Albert; Nzoue, Sita; Konan, Serges; Gnionsahe, Daze Apollinaire

    2015-04-01

    Viral infections are an important complication of transplantation. Polyomavirus are the commonest viruses that infect the renal allograft. Herpes virus nephropathy has also been described. In the past 15 years, adenovirus nephritis has emerged as a potentially life-threatening disease in renal transplant patients in developed countries. Most of the papers devoted to adenovirus nephritis are reported cases. The fate of such patients in resources-limited countries is not known. Herein, we describe the clinical, biological and prognostic findings of a black African transplanted patient with adenoviral hemorrhagic cystitis. This case is the very first of its kind reported in black Africa in a setting of a start of a renal transplantation pilot project. The patient is a 54-year-old man admitted at the nephrology service for gross haematuria and fever occurred 1 month after kidney transplantation. The diagnosis of adenoviral hemorrhagic cystitis has been suspected because the patient has displayed recurrent conjunctivitis and gastroenteritis well before transplantation, which was then confirmed by the real-time polymerase chain reaction performed on the blood. Conservatory measures associated with immunosuppression reduction have permitted the discontinuation of haematuria. This case has been discussed in regard of the epidemiology, the diagnosis, the treatment, the evolution and the prognosis of the adenoviral infection in the renal transplant patient. A review of the literature has been performed subsequently. Copyright © 2015 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

  19. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation......, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge...

  20. Ectopic protein interactions within BRD4–chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma

    OpenAIRE

    Alekseyenko, Artyom A.; Walsh, Erica M.; Zee, Barry M.; Pakozdi, Tibor; Hsi, Peter; Lemieux, Madeleine E.; Dal Cin, Paola; Ince, Tan A.; Kharchenko, Peter V.; Kuroda, Mitzi I.; French, Christopher A.

    2017-01-01

    Chromatin factors generally act within large, multisubunit complexes; thus, identifying both their normal and aberrant interactors in cancer should provide important information regarding potential targets for therapeutic intervention. Here, we apply this principle to analysis of BRD4–NUT, a fusion oncoprotein that drives an aggressive subtype of squamous cell cancer. We identify ZNF532 as a prominent BRD4–NUT–interacting protein in an established NUT midline carcinoma patient cell line, and ...

  1. Clinical Implication of Elevated Human Cervical Cancer Oncogene-1 Expression in Esophageal Squamous Cell Carcinoma

    OpenAIRE

    Liu, Ying; Li, Ke; Ren, Zhonghai; Li, Shenglei; Zhang, Hongyan; Fan, Qingxia

    2012-01-01

    The human cervical cancer oncogene 1 (HCCR-1), a novel human oncoprotein, has been shown to be upregulated in various human tumors and plays a critical role in tumorigenesis and tumor progression. Here, the authors investigated HCCR-1 level in esophageal squamous cell carcinoma (ESCC) tissues and assessed the correlation between HCCR-1 level and prognosis of the patients with ESCC. HCCR-1 levels were investigated by immunohistochemistry, in situ hybridization, real-time quantit...

  2. Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

    OpenAIRE

    Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

    2012-01-01

    Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chroma...

  3. Increased activity of c-Src and Csk in fibroblasts transformed by v-src oncogene

    Czech Academy of Sciences Publication Activity Database

    Tuháčková, Zdena; Vojtěchová, Martina; Hlaváček, Jan; Ruzzene, M.; Sovová, Vlasta; Pinna, L. A.

    2002-01-01

    Roč. 290, č. 42 (2002), s. 790-795 ISSN 0006-291X R&D Projects: GA ČR GV312/96/K205; GA ČR GA301/00/0269; GA MZd NC5428 Institutional research plan: CEZ:AV0Z5052915 Keywords : c-Src, v-Src oncoprotein * C-terminal c-Src kinase * Rous sarcoma virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.935, year: 2002

  4. Directional Migration in Esophageal Squamous Cell Carcinoma (ESCC) is Epigenetically Regulated by SET Nuclear Oncogene, a Member of the Inhibitor of Histone Acetyltransferase Complex

    OpenAIRE

    Xiang Yuan; Xinshuai Wang; Bianli Gu; Yingjian Ma; Yiwen Liu; Man Sun; Jinyu Kong; Wei Sun; Huizhi Wang; Fuyou Zhou; Shegan Gao

    2017-01-01

    Directional cell migration is of fundamental importance to a variety of biological events, including metastasis of malignant cells. Herein, we specifically investigated SET oncoprotein, a subunit of the recently identified inhibitor of acetyltransferases (INHAT) complex and identified its role in the establishment of front–rear cell polarity and directional migration in Esophageal Squamous Cell Carcinoma (ESCC). We further define the molecular circuits that govern these processes by showing t...

  5. Evasion of host immune defenses by human papillomavirus.

    Science.gov (United States)

    Westrich, Joseph A; Warren, Cody J; Pyeon, Dohun

    2017-03-02

    A majority of human papillomavirus (HPV) infections are asymptomatic and self-resolving in the absence of medical interventions. Various innate and adaptive immune responses, as well as physical barriers, have been implicated in controlling early HPV infections. However, if HPV overcomes these host immune defenses and establishes persistence in basal keratinocytes, it becomes very difficult for the host to eliminate the infection. The HPV oncoproteins E5, E6, and E7 are important in regulating host immune responses. These oncoproteins dysregulate gene expression, protein-protein interactions, posttranslational modifications, and cellular trafficking of critical host immune modulators. In addition to the HPV oncoproteins, sequence variation and dinucleotide depletion in papillomavirus genomes has been suggested as an alternative strategy for evasion of host immune defenses. Since anti-HPV host immune responses are also considered to be important for antitumor immunity, immune dysregulation by HPV during virus persistence may contribute to immune suppression essential for HPV-associated cancer progression. Here, we discuss cellular pathways dysregulated by HPV that allow the virus to evade various host immune defenses. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Molecular Docking Explains Atomic Interaction between Plant-originated Ligands and Oncogenic E7 Protein of High Risk Human Papillomavirus Type 16

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2014-12-01

    Full Text Available Cervical cancer caused by Human papillomavirus (HPV is one of the leading causes of cancer mortality in women worldwide, particularly in the developing countries. In the last few decades, various compounds from plant origin such as Curcumin, Epigallocatechin gallate (EGCG, Jaceosidin, Resveratrol etc. have been used as anti cancer therapeutic agents. Different studies have shown these plant-originated compounds are able to suppress HPV infection. The E6 and E7 oncoproteins of high-risk HPV play a key role in HPV related cancers. In this study, we explored these ligands from plants origin against E7 oncoprotein of high risk HPV 16, which is known to inactivate tumor suppressor pRb protein. A robust homology model of HPV 16 E7 was built to foresee the interaction mechanism of E7 oncoprotein with these ligands using structure-based drug designing approach. Docking studies demonstrate the interaction of these ligands with pRb binding site of E7 protein by residues Tyr52, Asn53, Val55, Phe57, Cys59, Ser63, Thr64, Thr72, Arg77, Glu80 and Asp81 and help restoration of pRb functioning. This in silico based atomic interaction between these ligands and E7 protein may assist in validating the plant-originated ligands as effective drugs against HPV.

  7. Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

    Directory of Open Access Journals (Sweden)

    Minglong Liu

    Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

  8. PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Hyo-Sup Kim

    Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

  9. Advances in study of reporter gene imaging for monitoring gene therapy

    International Nuclear Information System (INIS)

    Mu Chuanjie; Zhou Jiwen

    2003-01-01

    To evaluate the efficiency of gene therapy, it is requisite to monitor localization and expression of the therapeutic gene in vivo. Monitoring expression of reporter gene using radionuclide reporter gene technique is the best method. Adenoviral vectors expressing reporter gene are constructed using gene fusion, bicistronic, double promoter or bidirectional transcriptional recombination techniques, and transferred into target cells and tissues, then injected radiolabeled reporter probes which couple to the reporter genes. The reporter genes can be imaged invasively, repeatedly, quantitatively with γ-camera, PET and SPECT. Recently, several reporter gene and reporter probe systems have been used in studies of gene therapy. The part of them has been used for clinic trials

  10. Breast Cancer Research Program (BCRP) - Predoctoral Traineeship - Elucidating the Role of the Type III Transforming Growth Factor-beta Receptor in Bone Morphogenetic Signaling in Breast Cancer

    Science.gov (United States)

    2008-03-01

    phosphorylation in the pancreatic cancer cell model, Panc -1 (data not shown). This data emphasize that TβRIII’s role in BMP signaling is likely to be cell... Panc -1 cells were adenovirally infected with TβRIII, followed by BMP-4-induced EMT. The cells were then plated in Matrigel invasion chambers. A...BMP-2, while loss of TβRIII in Panc -1 (pancreatic cancer cells) increased cell sensitivity to BMP-2 and had no effect on MDA-MB-231 (breast cancer

  11. Therapeutic Endoscopic Ultrasonography: Intratumoral Injection for Pancreatic Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Lawrence A. Shirley

    2013-01-01

    Full Text Available Pancreatic adenocarcinoma is an aggressive disease that has poor outcomes despite maximal traditional therapies. Thus, treatment of this cancer demands innovative strategies to be used in addition to standing therapies in order to provide new avenues of care. Here, we describe the technique of using endoscopic ultrasound in order to directly inject both novel and conventional therapies into pancreatic tumors. We detail the rationale behind this strategy and the many benefits it provides. We then describe our technique in detail, including our experience injecting the AdV-tk adenoviral vector to create an in situ vaccine effect.

  12. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

  13. Factors influencing recombinant adeno-associated virus production.

    Science.gov (United States)

    Salvetti, A; Orève, S; Chadeuf, G; Favre, D; Cherel, Y; Champion-Arnaud, P; David-Ameline, J; Moullier, P

    1998-03-20

    Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary

  14. Interference with Intraepithelial TNF-α Signaling Inhibits CD8+ T-Cell-Mediated Lung Injury in Influenza Infection

    OpenAIRE

    Srikiatkhachorn, Anon; Chintapalli, Jyothi; Liu, Jun; Jamaluddin, Mohammad; Harrod, Kevin S.; Whitsett, Jeffrey A.; Enelow, Richard I.; Ramana, Chilakamarti V.

    2010-01-01

    CD8+ T-cell-mediated pulmonary immunopathology in respiratory virus infection is mediated in large part by antigen-specific TNF-α expression by antiviral effector T cells, which results in epithelial chemokine expression and inflammatory infiltration of the lung. To further define the signaling events leading to lung epithelial chemokine production in response to CD8+ T-cell antigen recognition, we expressed the adenoviral 14.7K protein, a putative inhibitor of TNF-α signaling, in the distal ...

  15. Hematopoiesis stimulation test by interleukin 1α gene transfer in the Cynomolgus macaque: application to secondary medullary aplasia from an accidental irradiation

    International Nuclear Information System (INIS)

    De Revel, Th.

    2002-12-01

    After a description of the context of medullary aplasia (haematological radiobiology, radiation acute syndrome, therapeutic care), and an overview of knowledge about the interleukin-1 and medullary stroma cells, this research thesis aims at investigating therapeutic alternatives for radio-accidental aplasia. More precisely, it aims at defining means to get cytokines which are efficient for haematopoiesis. Interleukin-1 is chosen for its properties and tests are performed on a macaque with two approaches for gene transfer: an ex vivo transfer by retroviral vector enabling an integration in the target cell genome, and an in situ transfer by adeno-viral vector directly applied in the animal osseous medulla

  16. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang

    2013-01-01

    infection with lymphocytic choriomeningitis virus. We found that localized immunization with intermediate doses of Ad vector induce a moderate number of functional CD8 T cells, which qualitatively match those found in LCMV-infected mice. Numbers of these cells may be efficiently increased by additional...... adenoviral boosting and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad-vaccination will lead to even higher numbers of memory cells. In this case, the vaccination...

  17. Acetyltransferases and tumour suppression

    International Nuclear Information System (INIS)

    Phillips, A C; Vousden, Karen H

    2000-01-01

    The acetyltransferase p300 was first identified associated with the adenoviral transforming protein E1A, suggesting a potential role for p300 in the regulation of cell proliferation. Direct evidence demonstrating a role for p300 in human tumours was lacking until the recentl publication by Gayther et al, which strongly supports a role for p300 as a tumour suppressor. The authors identify truncating mutations associated with the loss or mutation of the second allele in both tumour samples and cell lines, suggesting that loss of p300 may play a role in the development of a subset of human cancers

  18. VEGFR1-mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy

    DEFF Research Database (Denmark)

    Cao, Renhai; Xue, Yuan; Hedlund, Eva-Maria

    2010-01-01

    . Moreover, blockade of VEGFR1 but not VEGFR2 significantly restores pericyte saturation in mature retinal vessels. Our findings link VEGF and PlGF to cancer-associated retinopathy, reveal the molecular mechanisms of VEGFR1 ligand-mediated retinopathy, and define VEGFR1 as an important target......, and adenoviral vectors ablates pericytes from the mature retinal vasculature through the VEGF receptor 1 (VEGFR1)-mediated signaling pathway, leading to increased vascular leakage. In contrast, we demonstrate VEGF receptor 2 (VEGFR2) is primarily expressed in nonvascular photoreceptors and ganglion cells...

  19. Effect of HIV-1 envelope cytoplasmic tail on adenovirus primed virus encoded virus-like particle immunizations

    DEFF Research Database (Denmark)

    Andersson, Anne Marie C; Ragonnaud, Emeline; Seaton, Kelly E.

    2016-01-01

    were found between the different priming regimens as both induced high titered tier 1 neutralizing antibodies, but no tier 2 antibodies, possibly reflecting the similar presentation of trimer specific antibody epitopes. The described vaccine regimens provide insight into the effects of the HIV-1 Env......The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env...

  20. Adenovirus Protein E4-ORF1 activation of PI3 kinase reveals differential regulation of downstream effector pathways in adipocytes

    OpenAIRE

    Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K.; McGraw, Timothy E.

    2016-01-01

    Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but...

  1. Breadth of T cell responses after immunization with adenovirus vectors encoding ancestral antigens or polyvalent papillomavirus antigens

    DEFF Research Database (Denmark)

    Ragonnaud, Emeline; Pedersen, Anders Gorm; Holst, Peter Johannes

    2017-01-01

    to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...... circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did...

  2. Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

    Directory of Open Access Journals (Sweden)

    Arnouk Hilal

    2009-08-01

    Full Text Available Abstract Background Infection with high-risk type human papilloma viruses (HPVs is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE. The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23% of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2% were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1; and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27. Conclusion This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

  3. Merkel Cell Carcinomas Arising in Autoimmune Disease Affected Patients Treated with Biologic Drugs, Including Anti-TNF.

    Science.gov (United States)

    Rotondo, John Charles; Bononi, Ilaria; Puozzo, Andrea; Govoni, Marcello; Foschi, Valentina; Lanza, Giovanni; Gafà, Roberta; Gaboriaud, Pauline; Touzé, Françoise Antoine; Selvatici, Rita; Martini, Fernanda; Tognon, Mauro

    2017-07-15

    Purpose: The purpose of this investigation was to characterize Merkel cell carcinomas (MCC) arisen in patients affected by autoimmune diseases and treated with biologic drugs. Experimental Design: Serum samples from patients with MCC were analyzed for the presence and titer of antibodies against antigens of the oncogenic Merkel cell polyomavirus (MCPyV). IgG antibodies against the viral oncoproteins large T (LT) and small T (ST) antigens and the viral capsid protein-1 were analyzed by indirect ELISA. Viral antigens were recombinant LT/ST and virus-like particles (VLP), respectively. MCPyV DNA sequences were studied using PCR methods in MCC tissues and in peripheral blood mononuclear cells (PBMC). Immunohistochemical (IHC) analyses were carried out in MCC tissues to reveal MCPyV LT oncoprotein. Results: MCPyV DNA sequences identified in MCC tissues showed 100% homology with the European MKL-1 strain. PBMCs from patients tested MCPyV-negative. Viral DNA loads in the three MCC tissues were in the 0.1 to 30 copy/cell range. IgG antibodies against LT/ST were detected in patients 1 and 3, whereas patient 2 did not react to the MCPyV LT/ST antigen. Sera from the three patients with MCC contained IgG antibodies against MCPyV VP1. MCC tissues tested MCPyV LT-antigen-positive in IHC assays, with strong LT expression with diffuse nuclear localization. Normal tissues tested MCPyV LT-negative when employed as control. Conclusions: We investigated three new MCCs in patients affected by rheumatologic diseases treated with biologic drugs, including TNF. A possible cause-effect relationship between pharmacologic immunosuppressive treatment and MCC onset is suggested. Indeed, MCC is associated with MCPyV LT oncoprotein activity. Clin Cancer Res; 23(14); 3929-34. ©2017 AACR . ©2017 American Association for Cancer Research.

  4. The Potential Utility of Curcumin in the Treatment of HER-2-Overexpressed Breast Cancer: An In Vitro and In Vivo Comparison Study with Herceptin

    Directory of Open Access Journals (Sweden)

    Hung-Wen Lai

    2012-01-01

    Full Text Available HER-2 is an important oncoprotein overexpressed in about 15–25% of breast cancers. We hypothesized that the ability of curcumin to downregulate HER-2 oncoprotein and inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-κB activation may be important in the treatment of HER-2-overexpressed breast cancer. To examine the effect of curcumin on breast cancer cells, MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr (a herceptin resistant strain from SK-BR-3 cells were used for in vitro analysis. The in vivo effect of curcumin on HER-2-overexpressed breast cancer was investigated with the HER-2-overexpressed BT-474 xenograft model. Cell growth, cell cycle change, the antimobility effect, signal transduction, and xenograft volume analysis between groups treated with herceptin and/or curcumin were tested. Curcumin decreased the cell growth of various breast cancer cell lines (MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr. In Western blot analysis, the phosphorylation of Akt, MAPK, and expression of NF-κB were reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. When treated with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were decreased in both BT-474 and SK-BR-3-hr cells. In the BT-474 xenograft model, though not as much as herceptin, curcumin did effectively decrease the tumor size. The combination of curcumin with herceptin was not better than herceptin alone; however, the combination of taxol and curcumin had an antitumor effect comparable with taxol and herceptin. The results suggested that curcumin has potential as a treatment for HER-2-overexpressed breast cancer.

  5. An .I.ex vivo ./I.model to study v-Myb-induced leukemogenicity

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, Marta; Králová, Jarmila; Karafiát, Vít; Bartůněk, Petr; Dvořák, Michal

    2001-01-01

    Roč. 27, č. 2 (2001), s. 437-445 ISSN 1079-9796 R&D Projects: GA ČR GV301/98/K042; GA ČR GA204/00/0554; GA AV ČR IPP2052002 Grant - others:HHMI(US) 75195-540401 Institutional research plan: CEZ:AV0Z5052915 Keywords : v-Myb oncoprotein * PEST domain * leucine zipper Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.703, year: 2001

  6. E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

    International Nuclear Information System (INIS)

    Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha; Kongyingyoes, Bunkerd; Haonon, Ornuma; Boonmars, Thidarut; Kikawa, Satomi; Nakahara, Tomomi; Kiyono, Tohru; Ekalaksananan, Tipaya

    2016-01-01

    HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. - Highlights: • E6D25E HPV16 specifically modulates protein profile of human keratinocytes. • E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells. • E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.

  7. The cell biology of disease: Acute promyelocytic leukemia, arsenic, and PML bodies.

    Science.gov (United States)

    de Thé, Hugues; Le Bras, Morgane; Lallemand-Breitenbach, Valérie

    2012-07-09

    Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation.

  8. Template Based Design of Anti-Metastatic Drugs from the Active Conformation of Laminin Peptide II

    Science.gov (United States)

    2001-01-01

    affords lactam 10 as a 7 X=OH, Y=H Ph2BuSiO HH separable mixture of diastereomers rather 8 x = CI, Y NO2 9 10 than tricycle 11. Treatment of 10 with base...of mimotopes. explain the fact that pre- treatment of laminin with Amongst the phage that mimic the LBP൜ 9 peptide G in solution increases its...of ca6 - Colnagi, M. I. (1994). The simultaneous expression of integrin receptors and of mRNA encoding the puta- c-erbB-2 oncoprotein and laminin

  9. E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

    Energy Technology Data Exchange (ETDEWEB)

    Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha [Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 (Thailand); HPV & EBV and Carcinogenesis Research Group, Khon Kaen University (Thailand); Kongyingyoes, Bunkerd [Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 (Thailand); Haonon, Ornuma; Boonmars, Thidarut [Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 (Thailand); Kikawa, Satomi; Nakahara, Tomomi [Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045 (Japan); Kiyono, Tohru, E-mail: tkiyono@ncc.go.jp [Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045 (Japan); Ekalaksananan, Tipaya, E-mail: tipeka@kku.ac.th [Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 (Thailand); HPV & EBV and Carcinogenesis Research Group, Khon Kaen University (Thailand)

    2016-09-09

    HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. - Highlights: • E6D25E HPV16 specifically modulates protein profile of human keratinocytes. • E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells. • E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.

  10. Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma

    DEFF Research Database (Denmark)

    Helin, K; Holm, K; Niebuhr, A

    1997-01-01

    107, or p130 leads to growth arrest in the G1 phase of the cell cycle, and this arrest is abolished by complex formation with the adenovirus E1A, human papilloma virus E7, or simian virus 40 T oncoproteins. Inactivation of pRB by gross structural alterations or point mutations in the RB-1 gene has...... been described in a variety of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas. Despite the structural and functional similarity between pRB, p107, and p130, alterations in the latter two proteins have not been identified in human tumors. We have screened a panel...

  11. Protein mislocalization: mechanisms, functions and clinical applications in cancer

    Science.gov (United States)

    Wang, Xiaohong; Li, Shulin

    2014-01-01

    The changes from normal cells to cancer cells are primarily regulated by genome instability, which foster hallmark functions of cancer through multiple mechanisms including protein mislocalization. Mislocalization of these proteins, including oncoproteins, tumor suppressors, and other cancer-related proteins, can interfere with normal cellular function and cooperatively drive tumor development and metastasis. This review describes the cancer-related effects of protein subcellular mislocalization, the related mislocalization mechanisms, and the potential application of this knowledge to cancer diagnosis, prognosis, and therapy. PMID:24709009

  12. Gene therapy for carcinoma of the breast: Genetic ablation strategies

    International Nuclear Information System (INIS)

    Curiel, David T

    2000-01-01

    The gene therapy strategy of mutation compensation is designed to rectify the molecular lesions that are etiologic for neoplastic transformation. For dominant oncogenes, such approaches involve the functional knockout of the dysregulated cellular control pathways provoked by the overexpressed oncoprotein. On this basis, molecular interventions may be targeted to the transcriptional level of expression, via antisense or ribozymes, or post-transcriptionally, via intracellular single chain antibodies (intrabodies). For carcinoma of the breast, these approaches have been applied in the context of the disease linked oncogenes erbB-2 and cyclin D 1 , as well as the estrogen receptor. Neoplastic revision accomplished in modal systems has rationalized human trials on this basis

  13. Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

    Science.gov (United States)

    Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

    2015-10-26

    Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. MicroRNA in oral cancer research: future prospects.

    Science.gov (United States)

    Sarode, Sachin C; Sarode, Gargi S; Patil, Shankargouda

    2014-09-01

    MicroRNA (miRNA) and related therapeutic approaches hold great promise in the field of cancer managements. Various studies on epithelial malignancies have shown encouraging results on various fronts. Its association with invasion, tumor growth, epithelial mesenchymal transition (EMT), angiogenesis, cancer stem cells (CSCs), metastasis and refects the diversified role of miRNA. Moreover, miRNA plays an important role in determining the prognosis of the patients. MicroRNAs interactions with each other and with external factors [human papilloma virus (HPV) (like oncoproteins)] intrigue us to explore more deep into this fascinating world.(1.)

  15. Targeting HER 1 and 2 in breast cancer with lapatinib

    Directory of Open Access Journals (Sweden)

    Gerald M. Higa

    2011-12-01

    Full Text Available Numerous clinical trials support the biological relevance of the HER2 oncoprotein in breast cancer. In spite of improved outcomes, overexpression of the receptor is associated with increased risks of disease relapse, even in patients with early, and potentially curable, disease. Until recently, development of resistance to trastuzumab left patients with no therapeutic option other than specifically targeted HER2. This paper provides an in-depth review of lapatinib, a dual HER kinase inhibitor, as well as some insight into three HER family members, in breast cancer.

  16. Breast cancer metastasis driven by ErbB2 and 14-3-3ξ: A division of labor

    OpenAIRE

    Wang, Yingqun

    2010-01-01

    Metastasis remains the leading cause of cancer morbidity and mortality. ErbB2, a metastasis-promoting oncoprotein, is overexpressed in 50–60% of noninvasive ductal carcinoma in situ (DCIS). However, only 25% of invasive breast cancer (IBC) overexpress ErbB2, indicating that ErbB2 alone is not sufficient to drive metastasis and additional risk factors are necessary for the progression of ErbB2-overexpressing DCIS to IBC. A recent study published in Cancer Cell identified 14-3-3ξ as a risk fact...

  17. Epidemiology, biology and therapy of Merkel cell carcinoma: conclusions from the EU project IMMOMEC

    DEFF Research Database (Denmark)

    Becker, Juergen C.; Stang, Andreas; zur Hausen, Axel

    2018-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive, often lethal neuroendocrine cancer. Its carcinogenesis may be either caused by the clonal integration of the Merkel cell polyomavirus into the host genome or by UV-induced mutations. Notably, virally-encoded oncoproteins and UV-induced mutations...... knowledge on epidemiology, biology and therapy of MCC as conclusion of the project 'Immune Modulating strategies for treatment of Merkel Cell Carcinoma', which was funded over a 5-year period by the European Commission to investigate innovative immunotherapies for MCC....

  18. Chronic neutrophilic leukemia.

    Science.gov (United States)

    Bredeweg, Arthur; Burch, Micah; Krause, John R

    2018-01-01

    Chronic neutrophilic leukemia is a rare myeloproliferative disorder characterized by a sustained peripheral blood neutrophilia, absence of the BCR/ABL oncoprotein, bone marrow hypercellularity with less than 5% myeloblasts and normal neutrophil maturation, and no dysplasia. This leukemia has been associated with mutations in the colony-stimulating factor 3 receptor (CSF3R) that may activate this receptor, leading to the proliferation of neutrophils that are the hallmark of chronic neutrophilic leukemia. We present a case of chronic neutrophilic leukemia and discuss the criteria for diagnosis and the significance of mutations found in this leukemia.

  19. Getting genetic access to natural adenovirus genomes to explore vector diversity.

    Science.gov (United States)

    Zhang, Wenli; Ehrhardt, Anja

    2017-10-01

    Recombinant vectors based on the human adenovirus type 5 (HAdV5) have been developed and extensively used in preclinical and clinical studies for over 30 years. However, certain restrictions of HAdV5-based vectors have limited their clinical applications because they are rather inefficient in specifically transducing cells of therapeutic interest that lack the coxsackievirus and adenovirus receptor (CAR). Moreover, enhanced vector-associated toxicity and widespread preexisting immunity have been shown to significantly hamper the effectiveness of HAdV-5-mediated gene transfer. However, evolution of adenoviruses in the natural host is driving the generation of novel types with altered virulence, enhanced transmission, and altered tissue tropism. As a consequence, an increasing number of alternative adenovirus types were identified, which may represent a valuable resource for the development of novel vector types. Thus, researchers are focusing on the other naturally occurring adenovirus types, which are structurally similar but functionally different from HAdV5. To this end, several strategies have been devised for getting genetic access to adenovirus genomes, resulting in a new panel of adenoviral vectors. Importantly, these vectors were shown to have a host range different from HAdV5 and to escape the anti-HAdV5 immune response, thus underlining the great potential of this approach. In summary, this review provides a state-of-the-art overview of one essential step in adenoviral vector development.

  20. Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

    International Nuclear Information System (INIS)

    Han, Yu; Zhong, Cuiping; Hong, Liu; Wang, Ye; Qiao, Li; Qiu, Jianhua

    2009-01-01

    Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

  1. Tissue

    Directory of Open Access Journals (Sweden)

    David Morrissey

    2012-01-01

    Full Text Available Purpose. In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. Methods. Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. Results. In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. Conclusions. Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

  2. Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yu; Zhong, Cuiping [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Hong, Liu [First Division of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Wang, Ye; Qiao, Li [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China); Qiu, Jianhua, E-mail: qiujh@fmmu.edu.cn [Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi' an, 710032 Shaanxi Province (China)

    2009-12-18

    Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

  3. A novel gene therapy-based approach that selectively targets hypoxic regions within solid tumors

    International Nuclear Information System (INIS)

    Dougherty, S.T.; Dougherty, G.J.; Davis, P.D.

    2003-01-01

    There is compelling evidence that malignant cells present within the hypoxic regions that are commonly found within solid tumors contribute significantly to local recurrence following radiation therapy. We describe now a novel strategy designed to target such cells that exploits the differential production within hypoxic regions of the pro-angiogenic cytokine vascular endothelial cell growth factor (VEGF). Specifically, we have generated cDNA constructs that encode two distinct chimeric cell surface proteins that incorporate, respectively, the extracellular domains of the VEGF receptors Flk-1 or Flt-1, fused in frame to the membrane spanning and cytoplasmic domains of the pro-apoptotic protein Fas. Both chimeric proteins (Flk/Fas and Flt/Fas) appear stable and can be readily detected on the surface of transfected cells by Western blot and/or FACS analysis. Importantly, tumor cells expressing the chimeric proteins were rapidly killed in a dose-dependent fashion upon the addition of exogenous recombinant VEGF. Adenoviral vectors encoding Flk/Fas have been generated and shown to induce tumor cells to undergo apoptosis upon transfer to hypoxic conditions in vitro. This activity is dependent upon the endogenous production of VEGF. Studies are currently underway to test the ability of adenoviral Flk/Fas (Ad.Flk/Fas) to reduce tumor recurrence in vivo when used as an adjuvant therapy in conjunction with clinically relevant doses of ionizing radiation

  4. Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression

    International Nuclear Information System (INIS)

    Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian

    2007-01-01

    The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10 8 PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization

  5. Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging

    International Nuclear Information System (INIS)

    Maeda, Yasushi; Kimura, En; Uchida, Yuji; Nishida, Yasuto; Yamashita, Satoshi; Arima, Toshiyuki; Uchino, Makoto

    2003-01-01

    Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination

  6. Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells

    International Nuclear Information System (INIS)

    Zheng Changyu; O'Connell, Brian C.; Baum, Bruce J.

    2003-01-01

    Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event

  7. Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

    Science.gov (United States)

    Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

    2015-01-01

    Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

  8. Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

    Directory of Open Access Journals (Sweden)

    Dmitry Akhmedov

    Full Text Available The cAMP response element binding protein (CREB is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc. cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

  9. An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity

    Directory of Open Access Journals (Sweden)

    Wenli Zhang

    2017-05-01

    Full Text Available Adenoviruses (Ads are large human-pathogenic double-stranded DNA (dsDNA viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR and linear-circular homologous recombination (LCHR. We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS. We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

  10. Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats

    Directory of Open Access Journals (Sweden)

    Mikhail E. Sokolov

    2018-02-01

    Full Text Available Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF, glial cell-derived neurotrophic factor (GDNF, and neural cell adhesion molecule (NCAM successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.

  11. Dual oxidase maturation factor 1 (DUOXA1) overexpression increases reactive oxygen species production and inhibits murine muscle satellite cell differentiation.

    Science.gov (United States)

    Sandiford, Shelley D E; Kennedy, Karen A M; Xie, Xiaojun; Pickering, J Geoffrey; Li, Shawn S C

    2014-01-11

    Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate. To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype. This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.

  12. Neurotrophin gene therapy for sustained neural preservation after deafness.

    Science.gov (United States)

    Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Hume, Clifford R; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2012-01-01

    The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

  13. The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

    International Nuclear Information System (INIS)

    Yano, Fumiko; Kugimiya, Fumitaka; Ohba, Shinsuke; Ikeda, Toshiyuki; Chikuda, Hirotaka; Ogasawara, Toru; Ogata, Naoshi; Takato, Tsuyoshi; Nakamura, Kozo; Kawaguchi, Hiroshi; Chung, Ung-il

    2005-01-01

    To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (Canada) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of β-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

  14. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

    International Nuclear Information System (INIS)

    Campos, Samuel K.; Barry, Michael A.

    2006-01-01

    The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon

  15. Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells.

    Directory of Open Access Journals (Sweden)

    Sadhak Sengupta

    2011-03-01

    Full Text Available Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR. T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD.A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35-45% of splenic T cells were transduced by Ad-RGD.Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.

  16. Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

    Science.gov (United States)

    Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

    2015-11-01

    Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

  17. Overexpression of transforming growth factor-β1 in fetal monkey lung results in prenatal pulmonary fibrosis

    Science.gov (United States)

    Tarantal, A.F.; Chen, H.; Shi, T.T.; Lu, C-H.; Fang, A.B.; Buckley, S.; Kolb, M.; Gauldie, J.; Warburton, D.; Shi, W.

    2011-01-01

    Altered transforming growth factor (TGF)-β expression levels have been linked to a variety of human respiratory diseases, including bronchopulmonary dysplasia and pulmonary fibrosis. However, a causative role for aberrant TGF-β in neonatal lung diseases has not been defined in primates. Exogenous and transient TGF-β1 overexpression in fetal monkey lung was achieved by transabdominal ultrasound-guided fetal intrapulmonary injection of adenoviral vector expressing TGF-β1 at the second or third trimester of pregnancy. The lungs were then harvested near term, and fixed for histology and immunohistochemistry. Lung hypoplasia was observed where TGF-β1 was overexpressed during the second trimester. The most clearly marked phenotype consisted of severe pulmonary and pleural fibrosis, which was independent of the gestational time point when TGF-β1 was overexpressed. Increased cell proliferation, particularly in α-smooth muscle actin-positive myofibroblasts, was detected within the fibrotic foci. But epithelium to mesenchyme transdifferentiation was not detected. Massive collagen fibres were deposited on the inner and outer sides of the pleural membrane, with an intact elastin layer in the middle. This induced fibrotic pathology persisted even after adenoviral-mediated TGF-β1 overexpression was no longer evident. Therefore, overexpression of TGF-β1 within developing fetal monkey lung results in severe and progressive fibrosis in lung parenchyma and pleural membrane, in addition to pulmonary hypoplasia. PMID:20351039

  18. Autoinflammatory Reaction in Dogs Treated for Cancer via G6PD Inhibition

    Directory of Open Access Journals (Sweden)

    Jonathan W. Nyce

    2017-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is an oncoprotein that is overexpressed in cancer cells to provide the NADPH required for their increased anabolism. NADPH, sourced from G6PD fuels nucleotide biosynthesis, maintains redox potential of thioredoxin and glutathione and drives the mevalonate pathway that powers many of the basic mechanisms by which cancer cells escape host control. G6PD is thus a target for cancer treatment being addressed by many groups around the world. We have discovered that systemic inhibition of G6PD by high dose dehydroepiandrosterone (DHEA causes a severe autoinflammatory response in dogs, which does not occur in mice or rats. Since dogs more closely model the human adrenal androgen system than do common laboratory animals, this finding is relevant to the design of G6PD-inhibiting drugs for humans. The autoinflammatory reaction observed closely resembles mevalonate kinase deficiency (MKD, a rare autosomal recessive disease in humans characterized by recurrent febrile attacks, arthralgia, skin rash, and aphthous ulcers of mucocutaneous tissues. In a manner comparable to animal models of MKD, the reconstitution of protein geranylgeranylation blocked the autoinflammatory reaction caused by systemic G6PD inhibition. This autoinflammatory response to systemic G6PD inhibition represents an unexpected result that must be taken into consideration when targeting this oncoprotein.

  19. A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers.

    Science.gov (United States)

    Radaelli, Antonia; De Giuli Morghen, Carlo; Zanotto, Carlo; Pacchioni, Sole; Bissa, Massimiliano; Franconi, Rosella; Massa, Silvia; Paolini, Francesca; Muller, Antonio; Venuti, Aldo

    2012-12-01

    Development of effective therapeutic vaccines against human papilloma virus (HPV) infections remains a priority, considering the high number of new cases of cervical cancer each year by high-risk HPVs, in particular by HPV-16. Vaccines expressing the E7 oncoprotein, which is detectable in all HPV-positive pre-cancerous and cancer cells, might clear already established tumors and support the treatment of HPV-related lesions. In this study, DNA or fowlpox virus recombinants expressing the harmless variant E7GGG of the HPV-16 E7 oncoprotein (DNA(E7GGG) and FP(E7GGG)) were generated. Two immunization regimens were tested in a pre-clinical mouse model by homologous (FP/FP) or heterologous (DNA/FP) prime-boost protocols to evaluate the immune response and therapeutic efficacy of the proposed HPV-16 vaccine. Low levels of anti-E7-specific antibodies were elicited after immunization, and in vivo experiments resulted in a higher number of tumor-free mice after the heterologous immunization. These results establish a preliminary indication for therapy of HPV-related tumors by the combined use of DNA and avipox recombinants, which might represent safer immunogens than vaccinia-based vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

  1. SIAH1-induced p34SEI-1 polyubiquitination/degradation mediates p53 preferential vitamin C cytotoxicity.

    Science.gov (United States)

    Lee, Soonduck; Kim, Jinsun; Jung, Samil; Li, Chengping; Yang, Young; Kim, Keun Il; Lim, Jong-Seok; Kim, Yonghwan; Cheon, Choong-Il; Lee, Myeong-Sok

    2015-03-01

    Vitamin C is considered as an important anticancer therapeutic agent although this view is debatable. In this study, we introduce a physiological mechanism demonstrating how vitamin C exerts anticancer activity that induces cell cycle arrest and apoptosis. Our previous and current data reveal that p53 tumor suppressor is the prerequisite factor for stronger anticancer effects of vitamin C. In addition, vitamin C-mediated cancer cell cytotoxicity appears to be achieved at least partly through the downregulation of the p34SEI-1 oncoprotein. Our previous study showed that p34SEI-1 increases the survival of various types of cancer cells by inhibiting their apoptosis. Present data suggest that vitamin C treatment decreases the p34SEI-1 expression at the protein level and therefore alleviates its anti-apoptotic activity. Of note, SIAH1, E3 ubiquitin ligase, appears to be responsible for the p34SEI-1 polyubiquitination and its subsequent degradation, which is dependent on p53. In summary, vitamin C increases cancer cell death by inducing SIAH1-mediated polyubiquitination/degradation of the p34SEI-1 oncoprotein in a p53-dependent manner.

  2. Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

    Science.gov (United States)

    Minamitani, Takeharu; Ma, Yijie; Zhou, Hufeng; Kida, Hiroshi; Tsai, Chao-Yuan; Obana, Masanori; Okuzaki, Daisuke; Fujio, Yasushi; Kumanogoh, Atsushi; Zhao, Bo; Kikutani, Hitoshi; Kieff, Elliott; Gewurz, Benjamin E; Yasui, Teruhito

    2017-05-02

    Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

  3. Activation of the JNK pathway is essential for transformation by the Met oncogene.

    Science.gov (United States)

    Rodrigues, G A; Park, M; Schlessinger, J

    1997-05-15

    The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.

  4. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

    Science.gov (United States)

    Polte, T R; Hanks, S K

    1995-11-07

    The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

  5. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

    Science.gov (United States)

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-20

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  6. High Prevalence of Human Papillomavirus in Colorectal Cancer in Hispanics: A Case-Control Study

    Directory of Open Access Journals (Sweden)

    Raul D. Bernabe-Dones

    2016-01-01

    Full Text Available The role of Human Papillomavirus (HPV in colorectal carcinogenesis remains elusive. Based on the high incidence of HPV-associated malignancies among Puerto Rican Hispanics, this study aimed to assess the prevalence of HPV infection and viral integration in colorectal tissues in order to evaluate its putative role in colorectal cancer (CRC. In this case-control study, the prevalence of HPV infection in CRC (cases n = 45 and normal colon mucosa from cancer-free subjects (controls n = 36 was assessed by a nested PCR strategy. HPV-16 genotyping was performed in HPV-positive tissues and the physical status of the HPV-16 genome was determined by E2 detection. HPV was detected in 19 of 45 (42.2% CRC cases (mean age 61.1 ± 10.7 years, 24 males and in 1 of 36 (2.8% controls (mean age 60.9 ± 9.6 years, 24 males with an OR = 25.58 (95% CI 3.21 to 203.49. HPV-16 was detected in 63.2% of the HPV-positive colorectal tumors; genome integration was observed in all HPV-16 positive cases. This is the first report showing the high prevalence of HPV infections in Caribbean Hispanic colorectal tumors. Despite evidence of HPV integration into the host genome, further mechanistic analysis examining HPV oncoprotein expression and the putative role of these oncoproteins in colorectal carcinogenesis is warranted.

  7. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

    Science.gov (United States)

    Shaikh, Faraz; Sanehi, Parvish; Rawal, Rakesh

    2012-01-01

    Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. Abbreviations HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins. PMID:22829740

  8. Bcl-2 antisense therapy in B-cell malignancies.

    Science.gov (United States)

    Chanan-Khan, Asher

    2005-07-01

    Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.

  9. HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner.

    Science.gov (United States)

    Rabachini, Tatiana; Boccardo, Enrique; Andrade, Rubiana; Perez, Katia Regina; Nonogaki, Suely; Cuccovia, Iolanda Midea; Villa, Luisa Lina

    2018-04-27

    Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.

  10. BCL-2, in combination with MVP and IGF-1R expression, improves prediction of clinical outcome in complete response cervical carcinoma patients treated by radiochemotherapy.

    Science.gov (United States)

    Henríquez-Hernández, Luis Alberto; Lloret, Marta; Pinar, Beatriz; Bordón, Elisa; Rey, Agustín; Lubrano, Amina; Lara, Pedro Carlos

    2011-09-01

    To investigate whether BCL-2 expression would improve MVP/IGF-1R prediction of clinical outcome in cervix carcinoma patients treated by radiochemotherapy, and suggest possible mechanisms behind this effect. Fifty consecutive patients, who achieved complete response to treatment, from a whole series of 60 cases suffering from non-metastatic localized cervical carcinoma, were prospectively included in this study from July 1999 to December 2003. Follow-up was closed in January 2011. All patients received pelvic radiation (45-64.80 Gy in 1.8-2 Gy fractions) with concomitant cisplatin at 40 mg/m2/week doses followed by brachytherapy. Oncoprotein expression was studied by immunohistochemistry in paraffin-embedded tumour tissue. No relation was found between BCL-2 and clinicopathological variables. High MVP/IGF-1R/BCL-2 tumour expression was strongly related to poor local and regional disease-free survival (PMVP, and IGF-1R overexpression were related to poorer clinical outcome in cervical cancer patients who achieved clinical complete response to radiochemotherapy. The NHEJ repair protein Ku70/80 expression could be involved in the regulation of these oncoproteins. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    International Nuclear Information System (INIS)

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-01-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski

  12. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    International Nuclear Information System (INIS)

    Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

    2014-01-01

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects

  13. Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

    Directory of Open Access Journals (Sweden)

    Teresita Reiner

    Full Text Available Inhibition of the ubiquitin-proteasome system (UPS of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs, which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

  14. Characterization of HPV and host genome interactions in primary head and neck cancers

    Science.gov (United States)

    Parfenov, Michael; Pedamallu, Chandra Sekhar; Gehlenborg, Nils; Freeman, Samuel S.; Danilova, Ludmila; Bristow, Christopher A.; Lee, Semin; Hadjipanayis, Angela G.; Ivanova, Elena V.; Wilkerson, Matthew D.; Protopopov, Alexei; Yang, Lixing; Seth, Sahil; Song, Xingzhi; Tang, Jiabin; Ren, Xiaojia; Zhang, Jianhua; Pantazi, Angeliki; Santoso, Netty; Xu, Andrew W.; Mahadeshwar, Harshad; Wheeler, David A.; Haddad, Robert I.; Jung, Joonil; Ojesina, Akinyemi I.; Issaeva, Natalia; Yarbrough, Wendell G.; Hayes, D. Neil; Grandis, Jennifer R.; El-Naggar, Adel K.; Meyerson, Matthew; Park, Peter J.; Chin, Lynda; Seidman, J. G.; Hammerman, Peter S.; Kucherlapati, Raju; Ally, Adrian; Balasundaram, Miruna; Birol, Inanc; Bowlby, Reanne; Butterfield, Yaron S.N.; Carlsen, Rebecca; Cheng, Dean; Chu, Andy; Dhalla, Noreen; Guin, Ranabir; Holt, Robert A.; Jones, Steven J.M.; Lee, Darlene; Li, Haiyan I.; Marra, Marco A.; Mayo, Michael; Moore, Richard A.; Mungall, Andrew J.; Robertson, A. Gordon; Schein, Jacqueline E.; Sipahimalani, Payal; Tam, Angela; Thiessen, Nina; Wong, Tina; Protopopov, Alexei; Santoso, Netty; Lee, Semin; Parfenov, Michael; Zhang, Jianhua; Mahadeshwar, Harshad S.; Tang, Jiabin; Ren, Xiaojia; Seth, Sahil; Haseley, Psalm; Zeng, Dong; Yang, Lixing; Xu, Andrew W.; Song, Xingzhi; Pantazi, Angeliki; Bristow, Christopher; Hadjipanayis, Angela; Seidman, Jonathan; Chin, Lynda; Park, Peter J.; Kucherlapati, Raju; Akbani, Rehan; Casasent, Tod; Liu, Wenbin; Lu, Yiling; Mills, Gordon; Motter, Thomas; Weinstein, John; Diao, Lixia; Wang, Jing; Fan, You Hong; Liu, Jinze; Wang, Kai; Auman, J. Todd; Balu, Saianand; Bodenheimer, Tom; Buda, Elizabeth; Hayes, D. Neil; Hoadley, Katherine A.; Hoyle, Alan P.; Jefferys, Stuart R.; Jones, Corbin D.; Kimes, Patrick K.; Marron, J.S.; Meng, Shaowu; Mieczkowski, Piotr A.; Mose, Lisle E.; Parker, Joel S.; Perou, Charles M.; Prins, Jan F.; Roach, Jeffrey; Shi, Yan; Simons, Janae V.; Singh, Darshan; Soloway, Mathew G.; Tan, Donghui; Veluvolu, Umadevi; Walter, Vonn; Waring, Scot; Wilkerson, Matthew D.; Wu, Junyuan; Zhao, Ni; Cherniack, Andrew D.; Hammerman, Peter S.; Tward, Aaron D.; Pedamallu, Chandra Sekhar; Saksena, Gordon; Jung, Joonil; Ojesina, Akinyemi I.; Carter, Scott L.; Zack, Travis I.; Schumacher, Steven E.; Beroukhim, Rameen; Freeman, Samuel S.; Meyerson, Matthew; Cho, Juok; Chin, Lynda; Getz, Gad; Noble, Michael S.; DiCara, Daniel; Zhang, Hailei; Heiman, David I.; Gehlenborg, Nils; Voet, Doug; Lin, Pei; Frazer, Scott; Stojanov, Petar; Liu, Yingchun; Zou, Lihua; Kim, Jaegil; Lawrence, Michael S.; Sougnez, Carrie; Lichtenstein, Lee; Cibulskis, Kristian; Lander, Eric; Gabriel, Stacey B.; Muzny, Donna; Doddapaneni, HarshaVardhan; Kovar, Christie; Reid, Jeff; Morton, Donna; Han, Yi; Hale, Walker; Chao, Hsu; Chang, Kyle; Drummond, Jennifer A.; Gibbs, Richard A.; Kakkar, Nipun; Wheeler, David; Xi, Liu; Ciriello, Giovanni; Ladanyi, Marc; Lee, William; Ramirez, Ricardo; Sander, Chris; Shen, Ronglai; Sinha, Rileen; Weinhold, Nils; Taylor, Barry S.; Aksoy, B. Arman; Dresdner, Gideon; Gao, Jianjiong; Gross, Benjamin; Jacobsen, Anders; Reva, Boris; Schultz, Nikolaus; Sumer, S. Onur; Sun, Yichao; Chan, Timothy; Morris, Luc; Stuart, Joshua; Benz, Stephen; Ng, Sam; Benz, Christopher; Yau, Christina; Baylin, Stephen B.; Cope, Leslie; Danilova, Ludmila; Herman, James G.; Bootwalla, Moiz; Maglinte, Dennis T.; Laird, Peter W.; Triche, Timothy; Weisenberger, Daniel J.; Van Den Berg, David J.; Agrawal, Nishant; Bishop, Justin; Boutros, Paul C.; Bruce, Jeff P; Byers, Lauren Averett; Califano, Joseph; Carey, Thomas E.; Chen, Zhong; Cheng, Hui; Chiosea, Simion I.; Cohen, Ezra; Diergaarde, Brenda; Egloff, Ann Marie; El-Naggar, Adel K.; Ferris, Robert L.; Frederick, Mitchell J.; Grandis, Jennifer R.; Guo, Yan; Haddad, Robert I.; Hammerman, Peter S.; Harris, Thomas; Hayes, D. Neil; Hui, Angela BY; Lee, J. Jack; Lippman, Scott M.; Liu, Fei-Fei; McHugh, Jonathan B.; Myers, Jeff; Ng, Patrick Kwok Shing; Perez-Ordonez, Bayardo; Pickering, Curtis R.; Prystowsky, Michael; Romkes, Marjorie; Saleh, Anthony D.; Sartor, Maureen A.; Seethala, Raja; Seiwert, Tanguy Y.; Si, Han; Tward, Aaron D.; Van Waes, Carter; Waggott, Daryl M.; Wiznerowicz, Maciej; Yarbrough, Wendell; Zhang, Jiexin; Zuo, Zhixiang; Burnett, Ken; Crain, Daniel; Gardner, Johanna; Lau, Kevin; Mallery, David; Morris, Scott; Paulauskis, Joseph; Penny, Robert; Shelton, Candance; Shelton, Troy; Sherman, Mark; Yena, Peggy; Black, Aaron D.; Bowen, Jay; Frick, Jessica; Gastier-Foster, Julie M.; Harper, Hollie A.; Lichtenberg, Tara M.; Ramirez, Nilsa C.; Wise, Lisa; Zmuda, Erik; Baboud, Julien; Jensen, Mark A.; Kahn, Ari B.; Pihl, Todd D.; Pot, David A.; Srinivasan, Deepak; Walton, Jessica S.; Wan, Yunhu; Burton, Robert; Davidsen, Tanja; Demchok, John A.; Eley, Greg; Ferguson, Martin L.; Shaw, Kenna R. Mills; Ozenberger, Bradley A.; Sheth, Margi; Sofia, Heidi J.; Tarnuzzer, Roy; Wang, Zhining; Yang, Liming; Zenklusen, Jean Claude; Saller, Charles; Tarvin, Katherine; Chen, Chu; Bollag, Roni; Weinberger, Paul; Golusiński, Wojciech; Golusiński, Paweł; Ibbs, Matthiew; Korski, Konstanty; Mackiewicz, Andrzej; Suchorska, Wiktoria; Szybiak, Bartosz; Wiznerowicz, Maciej; Burnett, Ken; Curley, Erin; Gardner, Johanna; Mallery, David; Penny, Robert; Shelton, Troy; Yena, Peggy; Beard, Christina; Mitchell, Colleen; Sandusky, George; Agrawal, Nishant; Ahn, Julie; Bishop, Justin; Califano, Joseph; Khan, Zubair; Bruce, Jeff P; Hui, Angela BY; Irish, Jonathan; Liu, Fei-Fei; Perez-Ordonez, Bayardo; Waldron, John; Boutros, Paul C.; Waggott, Daryl M.; Myers, Jeff; Lippman, Scott M.; Egea, Sophie; Gomez-Fernandez, Carmen; Herbert, Lynn; Bradford, Carol R.; Carey, Thomas E.; Chepeha, Douglas B.; Haddad, Andrea S.; Jones, Tamara R.; Komarck, Christine M.; Malakh, Mayya; McHugh, Jonathan B.; Moyer, Jeffrey S.; Nguyen, Ariane; Peterson, Lisa A.; Prince, Mark E.; Rozek, Laura S.; Sartor, Maureen A.; Taylor, Evan G.; Walline, Heather M.; Wolf, Gregory T.; Boice, Lori; Chera, Bhishamjit S.; Funkhouser, William K.; Gulley, Margaret L.; Hackman, Trevor G.; Hayes, D. Neil; Hayward, Michele C.; Huang, Mei; Rathmell, W. Kimryn; Salazar, Ashley H.; Shockley, William W.; Shores, Carol G.; Thorne, Leigh; Weissler, Mark C.; Wrenn, Sylvia; Zanation, Adam M.; Chiosea, Simion I.; Diergaarde, Brenda; Egloff, Ann Marie; Ferris, Robert L.; Romkes, Marjorie; Seethala, Raja; Brown, Brandee T.; Guo, Yan; Pham, Michelle; Yarbrough, Wendell G.

    2014-01-01

    Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis. PMID:25313082

  15. Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

    Science.gov (United States)

    Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

    2001-03-15

    Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

  16. The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

    Science.gov (United States)

    Cheloni, Giulia; Tanturli, Michele

    2016-01-01

    Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets.

  17. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    Science.gov (United States)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  18. Soluble TGF-β type II receptor gene therapy reduces TGF-β activity in irradiated lung tissue and protects lungs from radiation-induced injury

    International Nuclear Information System (INIS)

    Vujaskovic, Z.; Rabbani, Z.; Zhang, X.; Samulski, T.V.; Li, C.-Y.; Anscher, M.S.

    2003-01-01

    Full text: The objective was to determine whether administration of recombinant human adenoviral vector carrying soluble TGF-β1 type II receptor (TβR-II) gene reduces availability of active TGFβ1 and protects lung from radiation-induced injury. Female Fisher-344 rats were randomized into four groups to receive: 1) Control 2) Adenoviral green fluorescent protein vector (AdGFP) alone 3) Radiation (RT) + Adenoviral vector with TGF-β1 type II receptor gene (AdexTβR-II-Fc) 4) RT alone. Animals were irradiated to right hemithorax using a single dose of 30 Gy. The packaging and production of a recombinant adenovirus carrying the fused human TβR-II-IgG1 Fc gene was achieved by use of the AdEasy system. The treatment vector AdexTbR-II-Fc (1.5*1010 PFU) and control vector AdGFP (1*109 PFU) were injected i.v. 24 hrs after RT. Respiratory rate was measured as an index of pulmonary function weekly for 5 weeks post RT. Structural damage was scored histologically. Immunohistochemistry was performed to identify activated macrophages. ELISA was used to quantify active TGF-β1 in tissue homogenate. Western blot was used to determine TβR-II expression in plasma and lung tissue. Animals receiving treatment vector AdexTbR-II-Fc have elevated plasma levels of soluble TβR-II at 24 and 48 hours after injection. In the RT+AdexTbR-II-Fc group, there was a significant reduction in respiratory rate (p = 0.002) at four weeks after treatment compared to RT alone group. Histology revealed a significant reduction in lung structural damage in animals receiving gene therapy after RT vs RT alone (p=0.0013). There was also a decrease in the number of activated macrophage (p= 0.02) in RT+AdexTbR-II-Fc group vs RT alone. The tissue protein expression of active TGF-β1 was significantly reduced in rats receiving RT+AdexTbR-II-Fc treatment (p<0.05). This study shows the ability of adenovirus mediated soluble TβR-II gene therapy to reduce tissue levels of active TGF-β1 and ameliorate radiation

  19. Biologic effect of exogenous wild p53 combined with irradiation on human melanoma cell lines with different p53 status

    International Nuclear Information System (INIS)

    Min Fengling; Zhang Hong; Li Wenjian; Liu Bing; Zhou Qingming; Duan Xin; Gao Qingxiang

    2007-01-01

    Objective: To investigate the effect of low dose irradiation on gene transfer efficiency and the effect of adenoviral-mediated exogenous P53 overexpression on apoptosis and radiosensitivity of radioresistant human melanoma cell lines A375(wild type p53)and WM983a(mutant type p53). Methods: Control vector, a replication deficient recombinant adenoviral vector containing a CMV promoter and green fluorescent protein (AdCMV-GFP), was used to transfect A375 cells and WM983a cells preirradiated with or without 1 Gy X-ray. The transduction efficiency of GFP gene was determined with fluorescence microscope directly. These two types of cells irradiated by 1 Gy X-ray were transfected with a replication deficient recombinant adenoviral vector carrying human wild p53 (AdCMV-p53), and mRNA level was detected by RT-PCR. The cell cycle delay and the expression of exogenous P53 were detected using flow cytometry (FCM) at different times after transfection. Tunel technique was used to detect cell apoptosis. The radiosensivity of A375 and WM983a cells after p53 transduction was analyzed by colony formation. Results: It is found that 1 Gy irradiation increased the gene transfection efficiency of A375 and WM983a cells. The expression of exogenous P53 was found to range from 60% to 80% among transfected cells during the first three days after transduction and then declined continuously down to the control level on day 10. G 1 cell cycle arrest was also observed after p53 gene transduction. WM983a cells transfected with p53 showed higher sensitivity to X-ray-induced cell killing than A375 cells. Conclusions: It is indicated that low dose of ionizing radiation can improve gene transfection efficiency of A375 and WM983a cells mediated by adenovirus vector. Althrough the overexpresion of exogenous p53 may not inhibit cell growth and induce apoptosis of melanoma cell line A375 and WM983a irt vitro, the two cell lines are much more sensitive to cell death induced by irradiation. It is

  20. Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

    Science.gov (United States)

    Maler, Mareike D; Nielsen, Peter J; Stichling, Nicole; Cohen, Idan; Ruzsics, Zsolt; Wood, Connor; Engelhard, Peggy; Suomalainen, Maarit; Gyory, Ildiko; Huber, Michael; Müller-Quernheim, Joachim; Schamel, Wolfgang