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

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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.

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

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

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

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

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.

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

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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.

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

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

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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.

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

Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description

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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.

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

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.

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

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

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.

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

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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.

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

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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.

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.

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

A Chlamydomonas-derived Human Papillomavirus 16 E7 vaccine induces specific tumor protection.

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Olivia C Demurtas

Full Text Available The E7 protein of the Human Papillomavirus (HPV type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG, under the control of the C. reinhardtii chloroplast psbD 5' UTR and the psbA 3' UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.The C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.

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.

Novel anti-metastatic action of cidofovir mediated by inhibition of E6/E7, CXCR4 and Rho/ROCK signaling in HPV tumor cells.

Directory of Open Access Journals (Sweden)

Abdessamad Amine

Full Text Available Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1alpha and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1alpha/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1alpha stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1 in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1alpha/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1alpha-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1alpha/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor. These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

Molecular Docking Explains Atomic Interaction between Plant-originated Ligands and Oncogenic E7 Protein of High Risk Human Papillomavirus Type 16

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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.

IFN-β antiproliferative effect and miRNA regulation in Human Papilloma Virus E6- and E7-transformed keratinocytes.

Science.gov (United States)

Chiantore, Maria Vincenza; Mangino, Giorgio; Iuliano, Marco; Zangrillo, Maria Simona; De Lillis, Ilaria; Vaccari, Gabriele; Accardi, Rosita; Tommasino, Massimo; Fiorucci, Gianna; Romeo, Giovanna

2017-01-01

Human Papilloma Viruses (HPVs) are the causative agents of cervical cancer although other types of cancers are associated with HPV infection. Type I Interferons can interfere with HPV E6- and/or E7-dependent transformation and can affect microRNA (miRNA) expression. Cancer cells show a specific pattern of miRNA expression and HPVs are able to modulate miRNAs expressed in infected cells. Keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38) were studied to analyze the involvement of HPV oncoproteins in the anti-proliferative activity of IFN-β. In view of our previous data showing senescence induction by the cytokine in K38 cells, we observe that IFN-β treatment leads to p53-indipendent apoptosis in K16 cells whereas induces senescence in K16 cells if E6 is silenced and p53 expression is restored. The levels of selected miRNAs, deregulated in K16 and K38 cells, can be modulated by IFN-β when E6 and E7 proteins of HPV-16, but not HPV-38, are expressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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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.

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.

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

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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.

The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells.

Science.gov (United States)

Javadi, Hamidreza; Lotfi, Abbas Sahebghadam; Hosseinkhani, Saman; Mehrani, Hossein; Amani, Jafar; Soheili, Zahra Soheila; Hojati, Zahra; Kamali, Mehdi

2018-06-06

In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.

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.

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....

Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach.

Directory of Open Access Journals (Sweden)

Christoph Jindra

Full Text Available Persistent infection with high-risk human papillomavirus (HPV types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c. prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.

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

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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.

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.

E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

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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.

Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions

Directory of Open Access Journals (Sweden)

Z.K. Tuong

2018-06-01

Full Text Available Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice, and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i the literature findings and ii the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines.

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

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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.

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.

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…

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.

Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice.

Science.gov (United States)

Poo, Haryoung; Pyo, Hyun-Mi; Lee, Tae-Young; Yoon, Sun-Woo; Lee, Jong-Soo; Kim, Chul-Joong; Sung, Moon-Hee; Lee, Seung-Hoon

2006-10-01

The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7-expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly-gamma-glutamic acid (gamma-PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei-PgsA-E7. E7-specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 +/- 1.8) and IFN-gamma secreting cells [510 +/- 86 spot-forming cells/10(6)cells] among splenocytes and increased IFN-gamma in supernatants of vaginal lymphocytes. Furthermore, in an E7-based mouse tumor model, animals receiving orally administered L. casei-PgsA-E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice. Copyright 2006 Wiley-Liss, Inc.

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.

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

Morphoproteomics, E6/E7 in-situ hybridization, and biomedical analytics define the etiopathogenesis of HPV-associated oropharyngeal carcinoma and provide targeted therapeutic options.

Science.gov (United States)

Brown, Robert E; Naqvi, Syed; McGuire, Mary F; Buryanek, Jamie; Karni, Ron J

2017-08-17

Human papillomavirus (HPV) has been identified as an etiopathogenetic factor in oropharyngeal squamous cell carcinoma. The HPV E6 and E7 oncogenes are instrumental in promoting proliferation and blocking differentiation leading to tumorigenesis. Although surgical intervention can remove such tumors, the potential for an etiologic field effect with recurrent disease is real. A downstream effector of E7 oncoprotein, enhancer of zeste homolog 2 (EZH2), is known to promote proliferation and to pose a block in differentiation and in turn, could lead to HPV-induced malignant transformation. However, the EZH2 pathway is amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease by inhibiting the incorporation of HPV into the genome. This is the first study using clinical specimens to demonstrate EZH2 protein expression in oropharyngeal carcinoma (OPC). The study included eight patients with oropharyngeal carcinoma, confirmed p16INK4a- positive by immunohistochemistry (IHC). The tissue expression of E6/E7 messenger RNA (mRNA) was measured by RNAscope® in-situ hybridization technology. Expression of EZH2, Ki-67, and mitotic indices were assessed by morphoproteomic analysis. Biomedical analytics expanded the results with data from Ingenuity Pathway Analysis (IPA) and KEGG databases to construct a molecular network pathway for further insights. Expression of E6 and E7 oncogenes in p16INK4a- positive oropharyngeal carcinoma was confirmed. EZH2 and its correlates, including elevated proliferation index (Ki-67) and mitotic progression were also present. Biomedical analytics validated the relationship between HPV- E6 and E7 and the expression of the EZH2 pathway. There is morphoproteomic and mRNA evidence of the association of p16INK4a-HPV infection with the E6 and E7 oncogenes and the expression of EZH2, Ki-67 and mitotic progression in oropharyngeal carcinoma. The molecular network biology was confirmed by

Gene silencing of HPV16 E6/E7 induced by promoter-targeting siRNA in SiHa cells

OpenAIRE

Hong, D; Lu, W; Ye, F; Hu, Y; Xie, X

2009-01-01

Background: Recently, transcriptional gene silencing induced by small interfering RNA (siRNA) was found in mammalian and human cells. However, previous studies focused on endogenous genes. Methods: In this study, we designed siRNA targeting the promoter of human papillomavirus 16 E6/E7 and transfected it into the cervical cancer cell line, SiHa. E6 and E7 mRNA and protein expression were detected in cells treated by promoter-targeting siRNA. Futhermore, cellular growth, proliferation, apoptos...

Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits.

Science.gov (United States)

Radaelli, Antonia; Pozzi, Eleana; Pacchioni, Sole; Zanotto, Carlo; Morghen, Carlo De Giuli

2010-04-21

Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP) recombinants separately expressing the HPV-16 E6 (FPE6) and E7 (FPE7) transgenes were used for priming, followed by E7 protein boosting. All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication.

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.

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

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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.

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

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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

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

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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

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

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Vera Lucia Nunes Pannain

2004-12-01

Full Text Available RACIONAL: A hepatocarcinogênese é um processo no qual as alterações genéticas e epigenéticas são bem conhecidas em modelos animais, mas carece de estudos no homem. OBJETIVOS: Analisar a freqüência das oncoproteínas p21ras, c-myc e p53 no carcinoma hepatocelular e no fígado não-neoplásico. Verificar ainda a associação destas oncoproteínas com os padrões e graus histológicos, assim como com as infecções pelos vírus das hepatites B e C. MÉTODOS: Foi analisada por método imunoistoquímico a detecção das oncoproteínas p21ras, c-myc e p53 em 47 casos de carcinoma hepatocelular e no tecido não-neoplásico circunjacente ao tumor (40 casos. RESULTADOS: As oncoproteínas p21ras, c-myc e p53 foram detectadas, respectivamente, em 44,7%, 53,2% e 36,2% dos casos de carcinoma hepatocelular. A imunorreatividade do p21ras e c-myc mostrou uma associação significativa. Contudo, não houve associação significativa entre a detecção do p21ras, c-myc e p53 com os diferentes graus e padrões histológicos, nem tampouco com as infecções pelos vírus das hepatites B e C. A mesma associação significativa entre o p21ras e c-myc foi encontrada no tecido não-neoplásico dos casos de cirrose em relação aos que não apresentaram cirrose, enquanto que o p53 foi negativo em todos os casos. CONCLUSÕES: A imunorreatividade das oncoproteínas p21ras, c-myc e p53 corrobora evidências prévias de sua detecção no carcinoma hepatocelular, o que sugere poder haver participação destas proteínas na hepatocarcinogênese humana. A significativa associação entre as proteínas p21ras, c-myc e p53 no carcinoma hepatocelular e na cirrose pode apontar uma interação entre as mesmas, sobretudo na hepatocarcinogênese pela via da cirrose.BACKGROUND: Genetic and epigenetic alterations have been described in animal hepatocarcinogenesis models but need to be studied in human being. AIMS: To assess the immunoreactivity of p21ras, c-myc and p53

Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits

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Pacchioni Sole

2010-04-01

Full Text Available Abstract Background Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Methods Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP recombinants separately expressing the HPV-16 E6 (FPE6 and E7 (FPE7 transgenes were used for priming, followed by E7 protein boosting. Results All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication.

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

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

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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.

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

Establishment and characterization of murine small cell lung carcinoma cell lines derived from HPV-16 E6/E7 transgenic mice.

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Carraresi, Laura; Martinelli, Rosanna; Vannoni, Alessandro; Riccio, Massimo; Dembic, Maja; Tripodi, Sergio; Cintorino, Marcella; Santi, Spartaco; Bigliardi, Elisa; Carmellini, Mario; Rossini, Mara

2006-01-08

We have established two murine cell lines derived from Small Cell Lung Carcinomas (SCLCs) developed by HPV-E6/E7 transgenic mice. These cells named PPAP-9 and PPAP-10 were isolated from mice bearing tumors, 9 and 10 months old, respectively. The cells, 5 microm in diameter, express HPV oncoproteins and sustain tumor formation after subcutaneous injection in syngenic mice. A detailed analysis indicated the epithelial origin and the neuroendocrine differentiation of these cells. We showed by confocal immunofluorescence the expression of the epithelial marker cytokeratin 5, whose gene promoter was used to direct the expression of HPV E6/E. Cells express several neuroendocrine markers such as CGRP, MAP-2, Ash1, CgrA, Scg2. The neuroendocrine differentiation of these cells was further confirmed by electron microscopy demonstrating neuropeptides secreting granules in their cytoplasm. Furthermore, in agreement with the altered expression observed in the majority of human SCLC we showed in these cells the absence of both p53 and pRB and a dramatic reduction in the expression of Caveolin-1.

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 ...

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.

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.

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

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.

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

Science.gov (United States)

You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

2014-03-01

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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....

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

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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.

FBXW7 Acts as an Independent Prognostic Marker and Inhibits Tumor Growth in Human Osteosarcoma

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Zhanchun Li

2015-01-01

Full Text Available F-box and WD repeat domain-containing 7 (FBXW7 is a potent tumor suppressor in human cancers including breast cancer, colorectal cancer, gastric cancer and hepatocellular carcinoma. In this study, we found that the expressions of FBXW7 protein and mRNA levels in osteosarcoma (OS cases were significantly lower than those in normal bone tissues. Clinical analysis indicated that FBXW7 was expressed at lower levels in OS patients with advanced clinical stage, high T classification and poor histological differentiation. Furthermore, we demonstrated that high expression of FBXW7 was correlated with a better 5-year survival of OS patients. Multivariate Cox regression analysis indicated that FBXW7 was an independent prognostic marker in OS. Our in vitro studies showed that FBXW7 overexpression inhibited cell cycle transition and cell proliferation, and promoted apoptosis in both U2OS and MG-63 cells. In a nude mouse xenograft model, FBXW7 overexpression slowed down tumor growth by inducing apoptosis and growth arrest. Mechanistically, FBXW7 inversely regulated oncoprotein c-Myc and cyclin E levels in both U2OS and MG-63 cells. Together these findings suggest that FBXW7 may serve as a prognostic biomarker and inhibit tumor progression by inducing apoptosis and growth arrest in OS.

HPV E6 and E7 in hypoxia mediated tumorigenesis in cervical epithelial cells

International Nuclear Information System (INIS)

Kim, Charlotte Y.; Tsai, Mitchell; Graeber, Thomas G.; Peehl, Donna M.; Giaccia, Amato J.

1996-01-01

Objective: In our previous work, we found that hypoxia induces apoptosis in oncogenically transformed rodent cells and loss of the p53 tumor suppressor gene significantly reduces hypoxia induced cell death. In this report, we show that transformation of wild-type p53 expressing primary cervical epithelial cells with the E6 and E7 genes from high risk human papillomavirus (HPV) type 16 dramatically enhances their susceptibility to hypoxia induced apoptosis. Materials and Methods: Sub confluent primary normal human cervical epithelial cells and normal human fibroblasts were infected with retroviral vectors containing HPV16 E6 and E7 and the neomycin selectable marker using previously described techniques. Clones were selected and isolated in neomycin containing media. Exponentially growing cells were treated with hypoxia (0.02% O 2 ) using specially designed chambers, irradiated (800 cGy) using a cesium source, or grown under aerobic conditions (20% O 2 ) as a control. After treatment, cells were stained with Hoescht and propidium iodide and viewed with a fluorescent microscope for analysis of apoptotic cells. To determine increase in expression of p53, immuno blots were performed using whole cell extracts. Results: After a 48 hour exposure to hypoxic conditions, 40% of E6 and E7 transformed cervical cells exhibit morphologic features indicative of apoptosis, compared to 5% of untransformed cervical cells. Exposure of HPV E6 and E7 transformed cells to ionizing radiation, however, did not initiate apoptosis. Immunoblot assays show induction of p53 under hypoxic conditions but not by ionizing radiation, indicating that hypoxia is able to induce p53 in the presence of E6 and that hypoxia activates p53 by a pathway which is distinct from that of ionizing radiation. Furthermore, hypoxia did not induce apoptosis in normal human fibroblasts transformed with E6 and E7, suggesting that the cellular response to hypoxia is influenced by the cell type. Conclusion: These results

7-ketocholesterol induces apoptosis of MC3T3-E1 cells associated with reactive oxygen species generation, endoplasmic reticulum stress and caspase-3/7 dependent pathway

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Yuta Sato

2017-03-01

Full Text Available Type 2 diabetes mellitus (T2DM is associated with an increased risk of bone fractures without reduction of bone mineral density. The cholesterol oxide 7-ketocholesterol (7KCHO has been implicated in numerous diseases such as atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer, age-related macular degeneration and T2DM. In the present study, 7KCHO decreased the viability of MC3T3-E1 cells, increased reactive oxygen species (ROS production and apoptotic rate, and upregulated the caspase-3/7 pathway. Furthermore, these effects of 7KCHO were abolished by pre-incubation of the cells with N-acetylcysteine (NAC, an ROS inhibitor. Also, 7KCHO enhanced the mRNA expression of two endoplasmic reticulum (ER stress markers; CHOP and GRP78, in MC3T3-E1 cells. Pre-incubation of the cells with NAC suppressed the 7KCHO-induced upregulation of CHOP, but not GRP78. In conclusion, we demonstrated that 7KCHO induced apoptosis of MC3T3-E1 cells associated with ROS generation, ER stress, and caspase-3/7 activity, and the effects of 7KCHO were abolished by the ROS inhibitor NAC. These findings may provide new insight into the relationship between oxysterol and pathophysiology of osteoporosis seen in T2DM.

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

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Coccia Raffaella

2009-01-01

Full Text Available Abstract Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14 by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these

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.

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

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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.

Oncoprotein metastasis and its suppression revisited

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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.

[Experimental study on carcinogenesis by human papillomavirus type 8 E7 gene].

Science.gov (United States)

Nishikawa, T

1994-05-01

Human papillomavirus (HPV) 5 and HPV8 are often detected in skin cancers developed in patients suffering from epidermodysplasia verruciformis, as well as in skin cancers developed in immunosuppressed patients. In the present study, in order to examine the transforming activity of the HPV8E7 gene, the HPV8E7 and HPV8E6/E7 genes were cloned into the expression vector (pcD2-Y), under the SV40 enhancer/promoter to construct pcD2-8E7 and pcD2-8E6/E7, respectively. The E7 and E6/E7 genes of genital high-risk HPV16 were also cloned into pcD2-Y to construct pcD2-16E7 and pcD2-16E6/E7, respectively. They were tested for their ability to collaboratively transform primary rat embryo fibroblasts (REFs) with activated H-ras gene. Transfection experiments of REFs having an activated H-ras gene revealed that pcD2-8E7, as well as pcD2-16E7 and pcD2-16E6/E7, induced transformation of cells in G418-resistant colonies at efficiencies of 11.9%, 43.0% and 53.0%, respectively. Transformed cell lines induced by activated H-ras gene and pcD2-8E7 or pcD2-16E7 were named 8RE and 16RE cell lines, respectively. Tumor induction in syngeneic newborn rats by injected the 8RE cells was higher than that of the 16RE cells. In cytological and histological examination, the 8RE cell lines and their induced tumors were different from the 16RE cell lines and their induced tumors. The 8RE cell lines showed the characteristic transformation with efficient growth ability on plastic and colony formation in 0.3% soft agar. These results support the hypothesis that the HPV8E7 gene plays an important role in the carcinogenesis of skin cancers.

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

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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

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.

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

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.

Molecular and Cytological Comparisons of Chromosomes 7el₁, 7el₂, 7E(e), and 7E ⁱ Derived from Thinopyrum.

Science.gov (United States)

Guo, Jun; He, Fang; Cai, Jin-Jin; Wang, Hong-Wei; Li, An-Fei; Wang, Hong-Gang; Kong, Ling-Rang

2015-01-01

Thinopyrum chromosomes 7el1, 7el2, 7E(e), and 7E(i), homoeologous to group 7 chromosomes of common wheat (Triticum aestivum), were determined to have many useful agronomical traits for wheat improvement. To analyze the genetic relationships among the 4 Thinopyrum 7E chromosomes, the conserved orthologous set markers, genomic in situ hybridization (GISH), and meiotic chromosome pairing were used in this study. The unweighted pair-group method with arithmetical averages (UPGMA) analysis indicated that 7el1, derived from T. ponticum, and 7E(i), derived from T. intermedium, were the most closely related. 7el2, derived from T. ponticum, was relatively distant from the 7el1-7E(i) complex. While 7E(e), derived from T. elongatum, was more distantly related to 7el1, 7el2, and 7E(i). This is the first report showing that 7el1 and 7E(i) may be similar, which could be explained by the similar chromosome signal distribution revealed by GISH as well as UPGMA analysis revealed by both molecular markers and the highest frequency of meiotic pairing. The newly developed genome-specific molecular markers may be useful for marker-assisted selection of Lr19, Bdv3, and Fhblop. © 2015 S. Karger AG, Basel.

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...

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

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.

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.

Interferon-β induced microRNA-129-5p down-regulates HPV-18 E6 and E7 viral gene expression by targeting SP1 in cervical cancer cells.

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Jiarong Zhang

Full Text Available Infection by human papillomavirus (HPV can cause cervical intraepithelial neoplasia (CIN and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.

High field induced magnetic transitions in the Y0.7E r0.3F e2D4.2 deuteride

Science.gov (United States)

Paul-Boncour, V.; Guillot, M.; Isnard, O.; Hoser, A.

2017-09-01

The influence of the partial Er for Y substitution on the crystal structure and magnetic properties of YF e2D4.2 has been investigated by high field magnetization and neutron diffraction experiments. Y0.7E r0.3F e2D4.2 compound crystallizes in the same monoclinic structure as YF e2D4.2 described in P c (P1c1) space group with D atoms located in 18 different tetrahedral interstitial sites. A cell volume contraction of 0.6% is observed upon Er substitution, inducing large modification of the magnetic properties. Electronic effect of D insertion as well as lowering of crystal symmetry are important factors determining the magnetic properties of Fe sublattice, which evolves towards more delocalized behavior and modifying the Er-Fe exchange interactions. In the ground state, the Er and Fe moments are arranged ferrimagnetically within the plane perpendicular to the monoclinic b axis and with average moments mEr=6.4 (3 ) μBEr-1 and mFe=2.0 (1 ) μBFe-1 at 10 K. Upon heating, mEr decreases progressively until TEr=55 K . Between 55 K and 75 K, the Fe sublattice undergoes a first-order ferromagnetic-antiferromagnetic (FM-AFM) transition with a cell volume contraction due to the itinerant metamagnetic behavior of one Fe site. In the AFM structure, mFe decreases until the Néel temperature TN=125 K . At high field, two different types of field induced transitions are observed. The Er moments become parallel to the Fe one and saturates to the E r3 + free ion value, leading to an unusual field induced FM arrangement at a transition field BTrans of only 78 kG below 30 K. Then above TM0=66 K , an AFM-FM transition of the Fe sublattice, accompanied by a cell volume increase is observed. BTrans increases linearly versus temperature and with a larger d BTrans/d T slope than for YF e2D4.2 . This has been explained by the additional contribution of Er induced moments above BTrans.

Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells

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Zheng Hu

2014-01-01

Full Text Available High-risk human papillomavirus (HR-HPV has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR- associated protein system (CRISPR/Cas system, a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

Potent anti-tumor effect generated by a novel human papillomavirus (HPV antagonist peptide reactivating the pRb/E2F pathway.

Directory of Open Access Journals (Sweden)

Cai-ping Guo

Full Text Available Human papillomavirus type 16 (HPV16 E7 is a viral oncoprotein believed to play a major role in cervical cancer. In this study, an antagonist peptide against HPV16E7 protein was first identified from screening the c7c phage display peptide library. The binding specificity and affinity of the selected peptide to HPV16E7 were tested by competitive enzyme-linked immunosorbent assay (ELISA. The antagonist peptide showed obvious anti-tumor efficacy both in cell lines and animal tumor models. Significant cell proliferation inhibition with high specificity was noted when HPV16-positive cells were treated with the peptide. This anti-tumor efficacy was resulted from overriding the activities of HPV16E7 and reactivating the pRb/E2F pathway, as shown by a series of experiments. Flow cytometry analysis revealed that the selected peptide induced G1 arrest in a dose-dependent manner. Competitive ELISA, pull down, and Co-IP experiments indicated that the selected peptide disrupted the interaction between HPV16E7 and pRb proteins both in vitro and in vivo. Luciferase reporter assay verified that transcription activities of E2F were suppressed by the peptide through restoration of pRb. RT-PCR and Western blot revealed that it reduced cyclins A, D1, and E1 expression, and led to HPV16E7 protein degradation, but pRb protein stabilization. The current study suggests that this specific peptide may serve as a potential therapeutic agent for HPV16-positive cervical cancer.

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.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

Science.gov (United States)

Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

2015-10-27

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

The epithelial-mesenchymal transition induced by keratinocyte growth conditions is overcome by E6 and E7 from HPV16, but not HPV8 and HPV38: Characterization of global transcription profiles

International Nuclear Information System (INIS)

Azzimonti, Barbara; Dell'Oste, Valentina; Borgogna, Cinzia; Mondini, Michele; Gugliesi, Francesca; De Andrea, Marco; Chiorino, Giovanna; Scatolini, Maria; Ghimenti, Chiara; Landolfo, Santo; Gariglio, Marisa

2009-01-01

The aim of this study was to evaluate the growth properties of primary human keratinocytes expressing E6 and E7 proteins, which are from either the β- or α-genotypes, under different culture conditions. We demonstrated that keratinocytes expressing E6 and E7, from both HPV8 and 38, irreversibly underwent the epithelial-mesenchymal transition (EMT) when grown on plastic with FAD medium (F12/DMEM/5%FBS). Expression of E6/E7 from HPV16 was capable of fully overcoming the FAD-induced EMT. Immortalization was only observed in HPV16-transduced cell lines, while the more proliferating phenotype of both KerHPV8 and 38 was mainly related to FAD-induced EMT. Microarray analysis of exponentially growing cells identified 146 cellular genes that were differentially regulated in HPV16 compared to HPV8- and 38-transduced cells. A large accumulation of transcripts associated with epidermal development and differentiation was observed in HPV16-transduced cells, whereas transcripts of genes involved in the extracellular matrix, multicellular organismal processes, and inflammatory response were affected in HPV8 and 38-transduced cells.

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...

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells.

Science.gov (United States)

Hong, Wen-Xu; Yang, Liang; Chen, Moutong; Yang, Xifei; Ren, Xiaohu; Fang, Shisong; Ye, Jinbo; Huang, Haiyan; Peng, Chaoqiong; Zhou, Li; Huang, Xinfeng; Yang, Fan; Wu, Desheng; Zhuang, Zhixiong; Liu, Jianjun

2012-09-01

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

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

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 ...

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells

Energy Technology Data Exchange (ETDEWEB)

Hong, Wen-Xu; Yang, Liang; Chen, Moutong; Yang, Xifei; Ren, Xiaohu; Fang, Shisong; Ye, Jinbo; Huang, Haiyan; Peng, Chaoqiong; Zhou, Li; Huang, Xinfeng; Yang, Fan; Wu, Desheng; Zhuang, Zhixiong; Liu, Jianjun, E-mail: bio-research@hotmail.com

2012-09-01

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity. -- Highlights: ► Identify 62 SET/TAF-Iα-associated proteins in human L-02 cells ► Trichloroethylene (TCE) alters the interaction of SET with eEF1A1 and eEF1A2. ► TCE induces the translocation and up-regulation of SET. ► TCE induces the translocation and up-regulation of eEF1A.

Proteomic analysis of trichloroethylene-induced alterations in expression, distribution, and interactions of SET/TAF-Iα and two SET/TAF-Iα-binding proteins, eEF1A1 and eEF1A2, in hepatic L-02 cells

International Nuclear Information System (INIS)

Hong, Wen-Xu; Yang, Liang; Chen, Moutong; Yang, Xifei; Ren, Xiaohu; Fang, Shisong; Ye, Jinbo; Huang, Haiyan; Peng, Chaoqiong; Zhou, Li; Huang, Xinfeng; Yang, Fan; Wu, Desheng; Zhuang, Zhixiong; Liu, Jianjun

2012-01-01

Emerging evidence indicates that trichloroethylene (TCE) exposure causes severe hepatotoxicity. However, the mechanisms of TCE hepatotoxicity remain unclear. Recently, we reported that TCE exposure up-regulated the expression of the oncoprotein SET/TAF-Iα and SET knockdown attenuated TCE-induced cytotoxicity in hepatic L-02 cells. To decipher the function of SET/TAF-Iα and its contributions to TCE-induced hepatotoxicity, we employed a proteomic analysis of SET/TAF-Iα with tandem affinity purification to identify SET/TAF-Iα-binding proteins. We identified 42 novel Gene Ontology co-annotated SET/TAF-Iα-binding proteins. The identifications of two of these proteins (eEF1A1, elongation factor 1-alpha 1; eEF1A2, elongation factor 1-alpha 2) were confirmed by Western blot analysis and co-immunoprecipitation (Co-IP). Furthermore, we analyzed the effects of TCE on the expression, distribution and interactions of eEF1A1, eEF1A2 and SET in L-02 cells. Western blot analysis reveals a significant up-regulation of eEF1A1, eEF1A2 and two isoforms of SET, and immunocytochemical analysis reveals that eEF1A1 and SET is redistributed by TCE. SET is redistributed from the nucleus to the cytoplasm, while eFE1A1 is translocated from the cytoplasm to the nucleus. Moreover, we find by Co-IP that TCE exposure significantly increases the interaction of SET with eEF1A2. Our data not only provide insights into the physiological functions of SET/TAF-Iα and complement the SET interaction networks, but also demonstrate that TCE exposure induces alterations in the expression, distribution and interactions of SET and its binding partners. These alterations may constitute the mechanisms of TCE cytotoxicity. -- Highlights: ► Identify 62 SET/TAF-Iα-associated proteins in human L-02 cells ► Trichloroethylene (TCE) alters the interaction of SET with eEF1A1 and eEF1A2. ► TCE induces the translocation and up-regulation of SET. ► TCE induces the translocation and up-regulation of eEF1A.

The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1

International Nuclear Information System (INIS)

Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Heng Koh, Choon; Zimmermann, Holger; Maolin Yang, Christopher; Guan Sim, Khe; I-Hong Hsu, Stephen; Bernard, Hans-Ulrich

2003-01-01

The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ('low-risk types'), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ('high-risk types'), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

Energy Technology Data Exchange (ETDEWEB)

Guo, Zheng [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Dadao Bei, Guangzhou 510515 (China); Zhou, Yuning [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Evers, B. Mark [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Wang, Qingding, E-mail: qingding.wang@uky.edu [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

International Nuclear Information System (INIS)

Guo, Zheng; Zhou, Yuning; Evers, B. Mark; Wang, Qingding

2012-01-01

Highlights: ► Rictor associates with FBXW7 to form an E3 complex. ► Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. ► Knockdown of rictor increases protein levels of c-Myc and cylin E. ► Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. ► Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor–FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

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

Modulation of Δ9-tetrahydrocannabinol-induced MCF-7 breast cancer cell growth by cyclooxygenase and aromatase

International Nuclear Information System (INIS)

Takeda, Shuso; Yamamoto, Ikuo; Watanabe, Kazuhito

2009-01-01

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC), a major constituent of marijuana, has been shown to stimulate the growth of MCF-7 breast cancer cells through cannabinoid receptor-independent signaling [Takeda, S., Yamaori, S., Motoya, E., Matsunaga, T., Kimura, T., Yamamoto, I., Watanabe, K., 2008. Δ 9 -Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling. Toxicology 245, 141-146]. Although the growth of MCF-7 cells is known to be stimulated by 17β-estradiol (E 2 ), the interaction of Δ 9 -THC and E 2 in MCF-7 cell growth is not fully clarified so far. In the present study, by using E 2 -sensitive MCF-7 cells that have expressed cyclooxygenase-2 (COX-2) and cytochrome P450 19 (aromatase), we studied whether or not COX-2 and aromatase are involved in Δ 9 -THC-mediated MCF-7 cell proliferation. It was shown that Δ 9 -THC-induced MCF-7 cell growth was inhibited by COX-2 inhibitors and was stimulated by arachidonic acid (a COX substrate). However, the growth of MCF-7 cells induced by Δ 9 -THC was not stimulated by PGE 2 , and the expression of aromatase was not affected by COX-2 inhibitors, arachidonic acid, and PGE 2 , suggesting that there is a disconnection between COX-2 (PGE 2 ) and aromatase in Δ 9 -THC-mediated MCF-7 cell proliferation. On the other hand, Δ 9 -THC-induced MCF-7 cell growth was elevated by two kinds of aromatase inhibitors. Taken together with the evidence that Δ 9 -THC-induced MCF-7 cell proliferation was interfered with testosterone (an aromatase substrate) and exogenously provided E 2 , it is suggested that (1) the growth stimulatory effects of Δ 9 -THC are mediated by the product(s) of COX-2 except for PGE 2 , (2) the action of Δ 9 -THC is modulated by E 2 , and (3) COX-2 and aromatase are individually engaged in the proliferation of MCF-7 cells induced by Δ 9 -THC.

HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

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Chong Wang

2016-04-01

Full Text Available The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

Science.gov (United States)

Wang, Chong; Long, Wenying; Peng, Chao; Hu, Lin; Zhang, Qiong; Wu, Ailing; Zhang, Xiaoqing; Duan, Xiaotao; Wong, Catherine C L; Tanaka, Yuetsu; Xia, Zongping

2016-04-01

The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

Detection of E6/E7 HPV oncogene transcripts as biomarker of cervical intaepithelial displasia

Directory of Open Access Journals (Sweden)

Mauro Carcheri

2009-09-01

Full Text Available It is widely accepted that only persistent infection with high risk types of Human Papillomavirus (HPV HR is a significant risk factor for the development of an invasive squamous cervical cancer. The overexpression of viral oncogenes E6/E7 of HPV is considered a necessary process for incurring in a malignant phenotype.A HPV infection can be identified by detection of HPV DNA in biological samples, but the DNAbased tests cannot delineate between transient or persistent and potentially transforming infection. Instead there is many evidence to suggest that detection of HPV gene expression may constitute a more specific approach to highlight a clinically significant infection. Especially seems that the detection of E6/E7 transcripts can be usefully used for identify the women with a persistent HPV infection that will can induce a future cervical cancer. The aim of our study is to investigate if the detection of oncogenic viral gene activity by detecting transcripts of the E6 and E7 genes can be most usefull of HPV-DNA test in the triage of ASCUS or low grade cervical lesions. Our results confirm that HPV E6/E7 mRNA test can be considered a promising method to stratify HPV positive women for risk of future high-grade cervical lesions or cervical intaepithelial neoplasia.

The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

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Orla Gannon

2011-12-01

Full Text Available The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8 appear to antagonize E2F-induced cell death. In this review we will discuss (i the potential role of E2Fs in UV-induced cell death and (ii the implications of this to the development of UV-induced cutaneous malignancies.

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

Expression of LIGHT/TNFSF14 Combined with Vaccination against Human Papillomavirus Type 16 E7 Induces Significant Tumor Regression

Science.gov (United States)

Kanodia, Shreya; Da Silva, Diane M.; Karamanukyan, Tigran; Bogaert, Lies; Fu, Yang-Xin; Kast, W. Martin

2010-01-01

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T-cells into the tumor. However, whether these infiltrating T-cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8+ T-cells that can be boosted using HPV16E6E7-Venezuelan Equine Encephalitis Virus Replicon Particles (HPV16-VRP) and that this combined therapy can eradicate HPV16-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of interferon gamma (IFNg) and chemottractant cytokines such as IL-1a, MIG and MIP-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8+ T-cells. Forced expression of LIGHT also results in the expansion of functional T-cells that recognize multiple tumor-antigens, including HPV16 E7, and these T-cells prevent the outgrowth of tumors upon secondary challenge. Subsequent boosting of E7-specific T-cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor, and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in pre-clinical studies and suggest that patients with HPV16+ tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination. PMID:20460520

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

Science.gov (United States)

Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

2003-09-01

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

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

HPV16-E7-Specific Activated CD8 T Cells in E7 Transgenic Skin and Skin Grafts

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Seyed Davoud Jazayeri

2017-05-01

Full Text Available Human papillomavirus (HPV 16 E7 (E7 protein expression in skin promotes epithelial hyperproliferation and transformation to malignancy. Grafts of murine skin expressing E7 protein as a transgene in keratinocytes are not rejected from immunocompetent recipients, whereas grafts expressing ovalbumin (OVA, with or without coexpression of E7 protein, are promptly rejected, demonstrating that E7-associated non-antigen-specific local immunosuppression is not a major determinant of lack of rejection of E7 transgenic skin. To determine whether failure of rejection of E7 skin grafts is due to failure to attract E7-specific effector T cells, E7- and OVA-specific effector CD8+ T cells, activated in vitro, were transferred to animals bearing E7 transgenic skin grafts. Three days after T cell transfer, E7-specific T cells were present in significantly greater numbers than OVA-specific T cells in the grafted skin on animals bearing recently placed or healed E7 grafts, without graft rejection, and also in the ear skin of E7 transgenic animals, without obvious pathology. E7 and OVA-specific T cells were present in lesser numbers in healed E7 grafts than in recently placed grafts and in lesser numbers in recently placed E7 transgenic epidermal grafts without E7-associated hyperproliferation, derived from E7 transgenic mice with a mutated retinoblastoma gene. These data demonstrate that effector T cells are to some extent attracted to E7 transgenic skin specifically by E7 expression, but in large measure non-specifically by the epithelial proliferation associated with E7 expression, and by the local inflammation produced by grafting. Failure of E7 graft rejection was observed despite trafficking of E7-specific effector T cells to E7-expressing epithelium, a finding of consequence for immunotherapy of HPV 16 E7-associated human cancers.

Protective effects of E838 on hematopoietic damage induced by radiaton

International Nuclear Information System (INIS)

Wang, Yueying; Liu, Qiang; Li, Deguan; Meng, Aimin; Wu, Hongying; Lu, Lu; Wang, Yong; Wang, Ruqin; Zhang, Liangan

2008-01-01

Full text: Purpose E838 was a new Ethinyl estradiol derivative with radioprotective property which could significantly elevate the irradiated mice survival rate of 30d. E838 also showed prevention and therapeutical effect in the leucopenia caused by radio-chemotherapy, but exhibit little estrogenic-like effects. In this study, we examined the protective effects of E838 on DNA damage and hematopoietic injury induced by irradiation in IRM-2 mice. Methods: IRM-2 mice were randomized into six groups: control, E838 5 mg/kg, E838 7.5 mg/kg, E838 10 mg/kg, ethinyl estradiol and nilestriol. After intraperitoneal injection 3 consecutive days, mice were exposed 1 Gy whole body γ-irradiation. Then the peripheral blood lymphocyte was collected to observe DNA double-strand breaks by neutral single cell gel electrophoresis (SCGE). Comet images were analyzed by CASP software. Two indexes were choiced, i.e. amount of DNA in the comet tail (tDNA %), Olive Tail Moment (OTM ). In the another experiment, treatment regimen same to the above experiment but the mice were exposed to 7.5 Gy whole body γ-irradiation. Bone marrow nucleated cell (BMC) per femur and endogenous spleen colony (CFU-S) were determined to evaluate the hematopoietic function at 9 days after irradiation. Results: tDNA % in different dose E838 (5, 7.5, 10 mg/kg) treated mice were decreased 67.6 %, 82.7 %, 74.9 % compared to the radiation control, respectively(P<0.001). The tDNA % reduction in 7.5mg/kg group were greater than those in ethinyl' estradiol (66.7 %) and nilestriol (66.7 %) treated mice significantly(P<0.01). OTM in E838 (5, 7.5, 10 mg/kg) treated irradiated mice were decreased 88.1 %, 95.3 %, 92.5 %(P<0.01), the OTM reduction in 5, 7.5mg/kg group were greater than those in ethinyl estradiol (86.3 %) and nilestriol(87.5 %) treated irradiated mice (P<0.01). BMC and CFU-S in E838 treated irradiated mice were 40 %∼60% higher than those in irradiated control mice (P<0.05∼0.01). Conclusion: The results

The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

Science.gov (United States)

Szalmás, Anita; Tomaić, Vjekoslav; Basukala, Om; Massimi, Paola; Mittal, Suruchi; Kónya, József; Banks, Lawrence

2017-04-01

Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays. IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor. Copyright © 2017 American Society for Microbiology.

Sensitization with 7S Globulins from Peanut, Hazelnut, Soy or Pea Induces IgE with Different Biological Activities Which Are Modified by Soy Tolerance

DEFF Research Database (Denmark)

Kroghsbo, Stine; Bøgh, Katrine Lindholm; Rigby, Neil M.

2011-01-01

, such as stability to digestion, have also been suggested. 7S globulins from peanut, hazelnut, soy, and pea were studied to determine whether related proteins would induce a similar sensitization when removed from their ‘normal’ matrix. Methods: Brown Norway rats (soy tolerant or nontolerant) were immunized i.p. 3......Background: It is not known why some foods sensitizing via the gastrointestinal tract are prevalent allergenic foods and others are not. Eating habits, processing, and the food matrix have been suggested to influence the allergenicity of a given food. Factors related to protein structure...... times with 100 μg purified peanut, hazelnut, soy, or pea 7S without adjuvant. Sera were analyzed for specific antibodies by different ELISAs (IgG1, IgG2a, and IgE), inhibition ELISA, and rat basophilic leukemia cell assay. Results: The 4 related 7S globulins induced a response with an almost identical...

IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex

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Tae Sik Goh

2017-01-01

Full Text Available IL-7 signaling via IL-7Rα and common γ-chain (γc is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα.

IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex

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Goh, Tae Sik; Jo, Yuna; Lee, Byunghyuk; Kim, Geona; Hwang, Hyunju; Ko, Eunhee; Kang, Seung Wan; Oh, Sae-Ock; Baek, Sun-Yong; Yoon, Sik; Lee, Jung Sub

2017-01-01

IL-7 signaling via IL-7Rα and common γ-chain (γc) is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα. PMID:28127156

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

Science.gov (United States)

Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2013-11-22

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

A Mouse Model of Hyperproliferative Human Epithelium Validated by Keratin Profiling Shows an Aberrant Cytoskeletal Response to Injury

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Samal Zhussupbekova

2016-07-01

Full Text Available A validated animal model would assist with research on the immunological consequences of the chronic expression of stress keratins KRT6, KRT16, and KRT17, as observed in human pre-malignant hyperproliferative epithelium. Here we examine keratin gene expression profile in skin from mice expressing the E7 oncoprotein of HPV16 (K14E7 demonstrating persistently hyperproliferative epithelium, in nontransgenic mouse skin, and in hyperproliferative actinic keratosis lesions from human skin. We demonstrate that K14E7 mouse skin overexpresses stress keratins in a similar manner to human actinic keratoses, that overexpression is a consequence of epithelial hyperproliferation induced by E7, and that overexpression further increases in response to injury. As stress keratins modify local immunity and epithelial cell function and differentiation, the K14E7 mouse model should permit study of how continued overexpression of stress keratins impacts on epithelial tumor development and on local innate and adaptive immunity.

HPV16-E7 expression in squamous epithelium creates a local immune suppressive environment via CCL2- and CCL5- mediated recruitment of mast cells.

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Anne-Sophie Bergot

2014-10-01

Full Text Available Human Papillomavirus (HPV 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.

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

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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.

Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice

International Nuclear Information System (INIS)

Whitehead, Mark; Öhlschläger, Peter; Almajhdi, Fahad N; Alloza, Leonor; Marzábal, Pablo; Meyers, Ann E; Hitzeroth, Inga I; Rybicki, Edward P

2014-01-01

Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

International Nuclear Information System (INIS)

Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

2009-01-01

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

Science.gov (United States)

Song, Shiyu; Pitot, Henry C.; Lambert, Paul F.

1999-01-01

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy. PMID:10364340

Alpha 7 nicotinic acetylcholine receptor-mediated protection against ethanol-induced neurotoxicity.

Science.gov (United States)

de Fiebre, NancyEllen C; de Fiebre, Christopher M

2003-11-01

The alpha(7)-selective nicotinic partial agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB) was examined for its ability to modulate ethanol-induced neurotoxicity in primary cultures of rat neurons. Primary cultures of hippocampal neurons were established from Long-Evans, embryonic day (E)-18 rat fetuses and maintained for 7 days. Ethanol (0-150 mM), DMXB (0-56 microM), or both were subsequently co-applied to cultures. Ethanol was added two additional times to the cultures to compensate for evaporation. After 5 days, neuronal viability was assessed with the MTT cell proliferation assay. Results demonstrated that ethanol reduces neuronal viability in a concentration-dependent fashion and that DMXB protects against this ethanol-induced neurotoxicity, also in a concentration-dependent fashion. These results support the suggestion that nicotinic partial agonists may be useful in treating binge drinking-induced neurotoxicity and may provide clues as to why heavy drinkers are usually smokers.

E7(7) constraints on counterterms in N=8 supergravity

International Nuclear Information System (INIS)

Beisert, N.; Elvang, H.; Freedman, D.; Kiermaier, M.; Morales, A.; Stieberger, S.

2010-01-01

We prove by explicit computation that 6-point matrix elements of D 4 R 4 and D 6 R 4 in N=8 supergravity have non-vanishing single-soft scalar limits, and therefore these operators violate the continuous E 7(7) symmetry. The soft limits precisely match automorphism constraints. Together with previous results for R 4 , this provides a direct proof that no E 7(7) -invariant candidate counterterm exists below 7-loop order. At 7-loops, we characterize the infinite tower of independent supersymmetric operators D 4 R 6 , R 8 , φ 2 R 8 , … with n>4 fields and prove that they all violate E 7(7) symmetry. This means that the 4-graviton amplitude determines whether or not the theory is finite at 7-loop order. We show that the corresponding candidate counterterm D 8 R 4 has a non-linear supersymmetrization such that its single- and double-soft scalar limits are compatible with E 7(7) up to and including 6-points. At loop orders 7,8,9 we provide an exhaustive account of all independent candidate counterterms with up to 16,14,12 fields, respectively, together with their potential single-soft scalar limits.

Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

Science.gov (United States)

Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

2015-04-01

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

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

Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

Science.gov (United States)

Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

2013-06-01

In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

Science.gov (United States)

Di Bonito, Paola; Ridolfi, Barbara; Columba-Cabezas, Sandra; Giovannelli, Andrea; Chiozzini, Chiara; Manfredi, Francesco; Anticoli, Simona; Arenaccio, Claudia; Federico, Maurizio

2015-01-01

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity. PMID:25760140

HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

Directory of Open Access Journals (Sweden)

Paola Di Bonito

2015-03-01

Full Text Available We developed an innovative strategy to induce a cytotoxic T cell (CTL immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut, which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV-E7 with that of lentiviral virus-like particles (VLPs incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

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

Directory of Open Access Journals (Sweden)

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

Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

International Nuclear Information System (INIS)

Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

2009-01-01

Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

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Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells

International Nuclear Information System (INIS)

Cherukuri, Durga Prasad; Chen, Xiao B.O.; Goulet, Anne-Christine; Young, Robert N.; Han, Yongxin; Heimark, Ronald L.; Regan, John W.; Meuillet, Emmanuelle; Nelson, Mark A.

2007-01-01

Accumulating evidence indicates that elevated levels of prostaglandin E 2 (PGE 2 ) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE 2 exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE 2 to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE 2 -induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE 2 induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE 2 treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE 2 driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE 2 in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer

[HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

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Xu, Yan; Cheng, Hao; Zhao, Ke-Jia; Zhu, Ke-Jian; Zhang, Xing

2007-11-01

This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.

BAG3 down-modulation sensitizes HPV18(+) HeLa cells to PEITC-induced apoptosis and restores p53.

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Cotugno, Roberta; Basile, Anna; Romano, Elena; Gallotta, Dario; Belisario, Maria Antonietta

2014-11-28

BAG3 is a multi-functional component of tumor cell pro-survival machinery, and its biological functions have been largely associated to proteasome system. Here, we show that BAG3 down-modulation resulted in reduced cell viability and enhanced PEITC-induced apoptosis largely more extensively in HeLa (HPV18(+)) rather than in C33A (HPV(-)) cervical carcinoma cell lines. Moreover, we demonstrate that BAG3 suppression led to a decrease of viral E6 oncoprotein and a concomitant recovery of p53 tumor suppressor, the best recognized target of E6 for proteasome degradation. E6 and p53 expression were modulated at protein level, since their respective mRNAs were unaffected. Taken together our findings reveal a novel role for BAG3 as host protein contributing to HPV18 E6-activated pro-survival strategies, and suggest a possible relevance of its expression levels in drug/radiotherapy-resistance of HPV18-bearing cervical carcinomas. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Activities of E7 promoters in the human papillomavirus type 16 genome during cell differentiation

DEFF Research Database (Denmark)

Hansen, Christina Neigaard; Nielsen, Lone; Norrild, Bodil

2010-01-01

Worldwide, one of the most common cancer forms diagnosed in women is cervical cancer induced by infections with high-risk human papillomaviruses (HPVs) with HPV type 16 (HPV-16) being the most frequently identified. The oncogenicity is caused mainly by expression of the oncogenes E6 and E7 leadin...

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...

Tinkertoys for the E 7 theory

Science.gov (United States)

Chacaltana, Oscar; Distler, Jacques; Trimm, Anderson; Zhu, Yinan

2018-05-01

We classify the class S theories of type E 7. These are four-dimensional N=2 superconformal field theories arising from the compactification of the E 7 (2, 0) theory on a punctured Riemann surface, C. The classification is given by listing all 3-punctured spheres ("fixtures"), and connecting cylinders, which can arise in a pants-decomposition of C. We find exactly 11,000 fixtures with three regular punctures, and an additional 48 with one "irregular puncture" (in the sense used in our previous works). To organize this large number of theories, we have created a web application at https://golem.ph.utexas.edu/class-S/E7/. Among these theories, we find 10 new ones with a simple exceptional global symmetry group, as well as a new rank-2 SCFT and several new rank-3 SCFTs. As an application, we study the strong-coupling limit of the E 7 gauge theory with 3 hypermultiplets in the 56. Using our results, we also verify recent conjectures that the T 2 compactification of certain 6 d (1, 0) theories can alternatively be realized in class S as fixtures in the E 7 or E 8 theories.

Interleukin-7 (IL-7) enhances class switching to IgE and IgG4 in the presence of T cells via IL-9 and sCD23.

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Jeannin, P; Delneste, Y; Lecoanet-Henchoz, S; Gretener, D; Bonnefoy, J Y

1998-02-15

Interleukin-7 (IL-7) is a B-cell growth factor produced by both bone marrow stroma cells and follicular dendritic cells (FDCs) located in primary lymphoid follicles and germinal centers. In this study, we have evaluated the role of IL-7 on human Ig class switching. IL-7 was added to peripheral blood mononuclear cells (PBMCs) or tonsillar B cells in the absence or presence of IL-4 and/or anti-CD40 monoclonal antibody (MoAb). Alone, IL-7 did not affect Ig production by PBMCs or by anti-CD40 MoAb-stimulated B cells. Rather, IL-7 potentiated IL-4-induced IgE and IgG4 production by PBMCs. In parallel, IgG3 production was also enhanced but to a lesser extent, whereas the production of the other isotypes was unaltered. The activity of IL-2, IL-9, or IL-15, which share usage of the common gamma chain for signaling, was also assessed. IL-9, like IL-7, potentiated mainly IgE and IgG4 production by IL-4-stimulated PBMCs. IL-15, in contrast, was ineffective, whereas IL-2 enhanced the production of all isotypes. More precisely, IL-7 potentiation of IgE and IgG4 production required the presence of T cells and was accompanied by an increase of the expression of two soluble molecules favoring preferentially IgE and IgG4 synthesis: CD23 (sCD23) and IL-9. Moreover, neutralizing anti-CD23 and anti-IL-9 antibodies partly inhibited the increase of IgE synthesis induced by IL-7. Thus, IL-7 produced locally in the germinal centers by FDCs may interact with T cells and potentiate human IgE and IgG4 switching by favoring IL-9 and sCD23 production.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

Science.gov (United States)

Albanese, Adriana; Sacerdoti, Flavia; Seyahian, E Abril; Amaral, Maria Marta; Fiorentino, Gabriela; Fernandez Brando, Romina; Vilte, Daniel A; Mercado, Elsa C; Palermo, Marina S; Cataldi, Angel; Zotta, Elsa; Ibarra, Cristina

2018-03-20

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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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.

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

Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade.

Directory of Open Access Journals (Sweden)

Elena Papaleo

Full Text Available The ubiquitin (Ub system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3. E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7, which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.

HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

Science.gov (United States)

Di Bonito, Paola; Ridolfi, Barbara; Columba-Cabezas, Sandra; Giovannelli, Andrea; Chiozzini, Chiara; Manfredi, Francesco; Anticoli, Simona; Arenaccio, Claudia; Federico, Maurizio

2015-03-09

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases.

Science.gov (United States)

Skeate, Joseph G; Woodham, Andrew W; Einstein, Mark H; Da Silva, Diane M; Kast, W Martin

2016-06-02

Carcinomas of the anogenital tract, in particular cervical cancer, remains one of the most common cancers in women, and represent the most frequent gynecological malignancies and the fourth leading cause of cancer death in women worldwide. Human papillomavirus (HPV)-induced lesions are immunologically distinct in that they express viral antigens, which are necessary to maintain the cancerous phenotype. The causal relationship between HPV infection and anogenital cancer has prompted substantial interest in the development of therapeutic vaccines against high-risk HPV types targeting the viral oncoproteins E6 and E7. This review will focus on the most recent clinical trials for immunotherapies for mucosal HPV-induced lesions as well as emerging therapeutic strategies that have been tested in pre-clinical models for HPV-induced diseases. Progress in peptide- and protein-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune checkpoint inhibition, immune response modifiers, and adoptive cell therapy for HPV will be discussed.

In Vitro and In Vivo Synergistic Therapeutic Effect of Cisplatin with Human Papillomavirus16 E6/E7 CRISPR/Cas9 on Cervical Cancer Cell Line

Directory of Open Access Journals (Sweden)

Shuai Zhen

2016-12-01

Full Text Available PURPOSE: Human papillomavirus (HPV type 16 is one of the major etiologic factors of cervical cancer. Our study aims to investigate the potentiality of the antiviral clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated Cas9 system (CRISPR/Cas9 targeting the E6 and E7 oncogenes of HPV16 as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP for cervical cancer. METHODS: Specifically, the therapeutic efficacy of combination of CDDP and HPV16 E6 + E7-CRISPR/Cas9 was assessed in cervical cancer cells and cervical cancer xenograft models. RESULTS: In vitro experiments showed that long-term exposure of SiHa cells to the HPV16 E6 + E7-CRISPR/Cas9 induced apoptosis, and its pro-apoptosis effect became more obvious when combined with CDDP. In vivo study found the efficacy of the combination of HPV16 E6 + E7-CRISPR/Cas9 and CDDP were superior to either of the treatments in term of apoptosis induction and metastasis inhibition. CONCLUSION: Collectively, our results suggested that HPV16 E6 + E7-CRISPR/Cas9 could be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Interferon-Tau has Antiproliferative effects, Represses the Expression of E6 and E7 Oncogenes, Induces Apoptosis in Cell Lines Transformed with HPV16 and Inhibits Tumor Growth In Vivo

Science.gov (United States)

Padilla-Quirarte, Herbey Oswaldo; Trejo-Moreno, Cesar; Fierros-Zarate, Geny; Castañeda, Jhoseline Carnalla; Palma-Irizarry, Marie; Hernández-Márquez, Eva; Burguete-Garcia, Ana Isabel; Peralta-Zaragoza, Oscar; Madrid-Marina, Vicente; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo

2016-01-01

Interferon tau (IFN-τ) is a promising alternative antiviral and immunotherapeutic agent in a wide variety of diseases including infectious, neurodegenerative, autoimmune and cancer due to its low toxicity in comparison with other type I interferon´s. The objective of our study was established the effect of the bovine IFN-τ on human (SiHa) and murine (BMK-16/myc) cells transformed with HPV 16 and evaluates the antitumor effect in a murine tumor model HPV 16 positive. We determine that bovine IFN-τ has antiproliferative effects, pro-apoptotic activity and induces repression of viral E6 and E7 oncogenes (time- and dose-dependent) on human and murine cells transformed with HPV 16 similar to the effects of IFN-β. However, IFN-τ induces greater antiproliferative effect, apoptosis and repression of both oncogenes in BMK-16/myc cells compared to SiHa cells. The differences were explained by the presence and abundance of the type I interferon receptor (IFNAR) in each cell line. On the other hand, we treated groups of tumor-bearing mice (HPV16 positive) with IFN-τ and showed the inhibition tumor growth effect in vivo. Our finding indicates that bovine IFN-τ may be a good candidate for immunotherapy against cervical cancer. PMID:27994659

A role of prostaglandin E/sub2/ in radiation-induced hyperthermia

International Nuclear Information System (INIS)

Cernak, I.; Stanimirovic, D.; Simovic, M.; Ivanovic, L.; Markovic, M.; Savic, J.

1989-01-01

Radiation-induced (6.7 Gy X-ray) changes in body temperature were investigated in rats exposed to: whole body irradiation pretreatment with indomethacin (5 mg/kg b.w.) 30 min before whole body irradiation and irradiation of the body with protected head. Content of prostaglandin E 2 (PGE 3 ) was measured in the rat brain hypothalamic regions and compared to correspondent body temperature. Hypothalamic PGE 2 content strongly correlated with body temperature (r=0.79, p 2 in the hypothalamus is presumed as a putative mediator of radiation-induced changes in body temperature. (author). 8 refs.; 2 figs.; 1 tab

Monitoring HPV-16 E7 phosphorylation events

Energy Technology Data Exchange (ETDEWEB)

Nogueira, Marcela O.; Hošek, Tomáš; Calçada, Eduardo O.; Castiglia, Francesca [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy); Massimi, Paola; Banks, Lawrence [International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste (Italy); Felli, Isabella C., E-mail: felli@cerm.unifi.it [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy); Pierattelli, Roberta, E-mail: pierattelli@cerm.unifi.it [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy)

2017-03-15

HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

Science.gov (United States)

Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

2017-01-01

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

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.

Recombinant Chimpanzee Adenovirus Vaccine AdC7-M/E Protects against Zika Virus Infection and Testis Damage.

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Xu, Kun; Song, Yufeng; Dai, Lianpan; Zhang, Yongli; Lu, Xuancheng; Xie, Yijia; Zhang, Hangjie; Cheng, Tao; Wang, Qihui; Huang, Qingrui; Bi, Yuhai; Liu, William J; Liu, Wenjun; Li, Xiangdong; Qin, Chuan; Shi, Yi; Yan, Jinghua; Zhou, Dongming; Gao, George F

2018-03-15

The recent outbreak of Zika virus (ZIKV) has emerged as a global health concern. ZIKV can persist in human semen and be transmitted by sexual contact, as well as by mosquitoes, as seen for classical arboviruses. We along with others have previously demonstrated that ZIKV infection leads to testis damage and infertility in mouse models. So far, no prophylactics or therapeutics are available; therefore, vaccine development is urgently demanded. Recombinant chimpanzee adenovirus has been explored as the preferred vaccine vector for many pathogens due to the low preexisting immunity against the vector among the human population. Here, we developed a ZIKV vaccine based on recombinant chimpanzee adenovirus type 7 (AdC7) expressing ZIKV M/E glycoproteins. A single vaccination of AdC7-M/E was sufficient to elicit potent neutralizing antibodies and protective immunity against ZIKV in both immunocompetent and immunodeficient mice. Moreover, vaccinated mice rapidly developed neutralizing antibody with high titers within 1 week postvaccination, and the elicited antiserum could cross-neutralize heterologous ZIKV strains. Additionally, ZIKV M- and E-specific T cell responses were robustly induced by AdC7-M/E. Moreover, one-dose inoculation of AdC7-M/E conferred mouse sterilizing immunity to eliminate viremia and viral burden in tissues against ZIKV challenge. Further investigations showed that vaccination with AdC7-M/E completely protected against ZIKV-induced testicular damage. These data demonstrate that AdC7-M/E is highly effective and represents a promising vaccine candidate for ZIKV control. IMPORTANCE Zika virus (ZIKV) is a pathogenic flavivirus that causes severe clinical consequences, including congenital malformations in fetuses and Guillain-Barré syndrome in adults. Vaccine development is a high priority for ZIKV control. In this study, to avoid preexisting anti-vector immunity in humans, a rare serotype chimpanzee adenovirus (AdC7) expressing the ZIKV M/E

Oct4 is required ~E7.5 for proliferation in the primitive streak.

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Brian DeVeale

2013-11-01

Full Text Available Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

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Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

2018-05-01

Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Negative Regulation of the Stability and Tumor Suppressor Function of Fbw7 by the Pin1 Prolyl Isomerase

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Min, Sang-Hyun; Lau, Alan W.; Lee, Tae Ho; Inuzuka, Hiroyuki; Wei, Shuo; Huang, Pengyu; Shaik, Shavali; Lee, Daniel Yenhong; Finn, Greg; Balastik, Martin; Chen, Chun-Hau; Luo, Manli; Tron, Adriana E.; DeCaprio, James A.; Zhou, Xiao Zhen; Wei, Wenyi; Lu, Kun Ping

2012-01-01

SUMMARY Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy. PMID:22608923

Abortamento espontâneo e provocado: ansiedade, depressão e culpa Spountaneous and induced abortion: anxiety, depression and guilty

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Gláucia Rosana Guerra Benute

2009-01-01

Full Text Available OBJETIVOS: Caracterizar a população que sofreu abortamento; investigar a existência de ansiedade e depressão; verificar se existe ou não sentimento de culpa após o abortamento e comparar os resultados entre mulheres que sofreram abortamento espontâneo e as que provocaram-no. MÉTODOS: 50 mulheres com abortamento espontâneo e 50 com provocado foram entrevistadas 30 dias após o abortamento. Foi realizada entrevista com questões abertas e fechadas e aplicada a escala Hospital Anxiety and depression. RESULTADOS: As mulheres que viveram o abortamento espontâneo encontram-se mais culpadas (30% que as que provocaram-no (18%. No entanto, as mulheres que provocaram o abortamento encontraram-se mais ansiosas (média de 11 e mais deprimidas (média de 8,3 que as mulheres que viveram abortamento espontâneo (médias de 8,7 e 6,1; respectivamente, pBACKGROUND: Pregnancy has a symbolic meaning for each woman. It varies according to personality structure and is related to women's previous life experiences. OBJECTIVES: the aim was to characterize the women that suffered abortion, asking about anxiety and depression, looking for guilty feelings after abortion, and to compare results between women who suffered spontaneous abortion and those who had intentional abortion. METHODS: fifty women with spontaneous and fifty with induced abortion were interviewed 30 days after the procedure. A semistructured questionnaire with open and closed-end questions and Hospital Anxiety and Depression Scale were administered. RESULTS: woman who induced abortion revealed to be more anxious (mean 11 and depressed (mean 8.3 than woman with spontaneous abortion (means 8.7 and 6.1 respectively, p<0.05. CONCLUSIONS: women who presented induced abortion were more anxious and depressed, as shown by later life events, full of problematic feelings and the need fort sychological support.

Metaloproteinases 1 e 7 e câncer colorretal Metalloproteinases 1 and 7 and colorectal cancer

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Mário Jucá

2008-09-01

Full Text Available A metaloproteinase-1 (MMP-1 e a metaloproteinase-7 (MMP-7 são proteinases da matriz extracelular (MEC, zinco-dependentes, envolvidas no processo inicial da carcinogênese por permitirem a invasão tumoral na célula e promover o processo de metastatização. O polimorfismo dessas proteinases tem sido estudado recentemente com o objetivo de validar susa expressão e/ou atividade como marcador prognóstico. Evidências cumulativas revelam importante papel das MMP's 1 e 7 em diferentes fases da carcinogênese. A MMP-1 tem ação direta sobre a principal proteína da MEC, que é o colágeno do tecido intersticial conectivo. Sua expressão aumentada neste tecido pode indicar alto potencial de disseminação tumoral em diferentes tipos de câncer, incluindo o colorretal. A associação deste aumento da expressão também parece ser verdadeira para a MMP-7.The metalloproteinase-1 (MMP-1 and metalloproteinase-7 (MMP-7 are proteinases of the extracellular matrix (MEC, zinc-dependent, involved in the initial process of carcinogenesis, allowing the invasion by the tumor cell and promoting the process of metastasis. The polymorphism of these proteinases has been studied recently in order to validate its expression and / or activity as a marker prognosis. Evidence shows cumulative important role of MMPs 1 and 7 in different stages of carcinogenesis. The MMP-1 is direct action on the main protein of the MEC, which is the collagen of interstitial connective tissue. Its increased expression in this tissue may indicate high potential for spread in different tumor types of cancer, including colorectal. The association of this increase of expression also appears to be true for MMP-7.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

International Nuclear Information System (INIS)

Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

2008-01-01

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19 ARF induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53

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...

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

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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.

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

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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.

Artonin E induces p53-independent G1 cell cycle arrest and apoptosis through ROS-mediated mitochondrial pathway and livin suppression in MCF-7 cells

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Etti IC

2017-03-01

Full Text Available Imaobong Christopher Etti,1,2 Abdullah Rasedee,3 Najihah Mohd Hashim,4 Ahmad Bustamam Abdul,5 Arifah Kadir,6 Swee Keong Yeap,7 Peter Waziri,5 Ibrahim Malami,5 Kian Lam Lim,8 Christopher J Etti9 1Pharmacology and Toxicology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 2Department of Pharmacology and Toxicology, University of Uyo, Uyo, Nigeria; 3Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 4Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 5MAKNA-Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia; 6Department of Veterinary Preclinical Science, Universiti Putra Malaysia, Serdang, Malaysia; 7Laboratory of Vaccine and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia; 8Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Selangor, Malaysia; 9Department of Agricultural and Food Engineering, University of Uyo, Uyo, Nigeria Abstract: Artonin E is a prenylated flavonoid compound isolated from the stem bark of Artocarpus elasticus. This phytochemical has been previously reported to be drug-like with full compliance to Lipinski’s rule of five and good physicochemical properties when compared with 95% of orally available drugs. It has also been shown to possess unique medicinal properties that can be utilized in view of alleviating most human disease conditions. In this study, we investigated the cytotoxic mechanism of Artonin E in MCF-7 breast cancer cells, which has so far not been reported. In this context, Artonin E significantly suppressed the breast cancer cell’s viability while inducing apoptosis in a dose-dependent manner. This apoptosis induction was caspase dependent, and it is mediated mainly through the intrinsic pathway with the elevation of total reactive oxygen species

Transforming properties of Felis catus papillomavirus type 2 E6 and E7 putative oncogenes in vitro and their transcriptional activity in feline squamous cell carcinoma in vivo

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Altamura, Gennaro, E-mail: gennaro.altamura@unina.it [Department of Veterinary Medicine and Animal Productions, General Pathology and Pathological Anatomy Unit, University of Naples Federico II, Via Delpino 1, 80137 Naples (Italy); Corteggio, Annunziata, E-mail: ancorteg@unina.it [Department of Veterinary Medicine and Animal Productions, General Pathology and Pathological Anatomy Unit, University of Naples Federico II, Via Delpino 1, 80137 Naples (Italy); Pacini, Laura, E-mail: PaciniL@students.iarc.fr [Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon (France); Conte, Andrea, E-mail: andreaconte88@hotmail.it [Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples (Italy); Pierantoni, Giovanna Maria, E-mail: gmpieran@unina.it [Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples (Italy); Tommasino, Massimo, E-mail: tommasinom@iarc.fr [Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon (France); Accardi, Rosita, E-mail: accardir@iarc.fr [Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon (France); Borzacchiello, Giuseppe, E-mail: borzacch@unina.it [Department of Veterinary Medicine and Animal Productions, General Pathology and Pathological Anatomy Unit, University of Naples Federico II, Via Delpino 1, 80137 Naples (Italy)

2016-09-15

Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting in upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC. - Highlights: • FcaPV2 E6 binds to and deregulates feline p53 protein. • FcaPV2 E7 binds to and deregulates feline pRb protein. • FcaPV2 oncogenes inhibit UVB-induced apoptosis. • FcaPV2 E6E7 and E7 increase the lifespan of primary cells. • FcaPV2 E2, E6 and E7 are expressed at the mRNA level in feline SCC in vivo.

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

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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.

p53 Loss Synergizes with Estrogen and Papillomaviral Oncogenes to Induce Cervical and Breast Cancers

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Shai, Anny; Pitot, Henry C.; Lambert, Paul F.

2010-01-01

Whereas the tumor suppressor p53 gene is frequently mutated in most human cancers, this is not the case in human papillomavirus (HPV)-associated cancers, presumably because the viral E6 oncoprotein inactivates the p53 protein. The ability of E6 to transform cells in tissue culture and induce cancers in mice correlates in part with its ability to inactivate p53. In this study, we compared the expression of the HPV16 E6 oncogene to the conditional genetic disruption of p53 in the context of a mouse model for cervical cancer in which estrogen is a critical cofactor. Nearly all of the K14Crep53f/f mice treated with estrogen developed cervical cancer, a stark contrast to its complete absence in like-treated K14E6WTp53f/f mice, indicating that HPV16 E6 must only partially inactivate p53. p53-independent activities of E6 also contributed to carcinogenesis, but in the female reproductive tract, these activities were manifested only in the presence of the HPV16 E7 oncogene. Interestingly, treatment of K14Crep53f/f mice with estrogen also resulted in mammary tumors after only a short latency, many of which were positive for estrogen receptor α. The majority of these mammary tumors were of mixed cell types, suggestive of their originating from a multipotent progenitor. Furthermore, a subset of mammary tumors arising in the estrogen-treated, p53-deficient mammary glands exhibited evidence of an epithelial to mesenchymal transition. These data show the importance of the synergy between estrogen and p53 insufficiency in determining basic properties of carcinogenesis in hormone-responsive tissues, such as the breast and the reproductive tract. PMID:18413729

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.

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

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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.

Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

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Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

2015-01-01

E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

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Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

Rescue of p53 function by small-molecule RITA in cervical carcinoma by blocking E6-mediated degradation.

Science.gov (United States)

Zhao, Carolyn Ying; Szekely, Laszlo; Bao, Wenjie; Selivanova, Galina

2010-04-15

Proteasomal degradation of p53 by human papilloma virus (HPV) E6 oncoprotein plays a pivotal role in the survival of cervical carcinoma cells. Abrogation of HPV-E6-dependent p53 destruction can therefore be a good strategy to combat cervical carcinomas. Here, we show that a small-molecule reactivation of p53 and induction of tumor cell apoptosis (RITA) is able to induce the accumulation of p53 and rescue its tumor suppressor function in cells containing high-risk HPV16 and HPV18 by inhibiting HPV-E6-mediated proteasomal degradation. RITA blocks p53 ubiquitination by preventing p53 interaction with E6-associated protein, required for HPV-E6-mediated degradation. RITA activates the transcription of proapoptotic p53 targets Noxa, PUMA, and BAX, and repressed the expression of pro-proliferative factors CyclinB1, CDC2, and CDC25C, resulting in p53-dependent apoptosis and cell cycle arrest. Importantly, RITA showed substantial suppression of cervical carcinoma xenografts in vivo. These results provide a proof of principle for the treatment of cervical cancer in a p53-dependent manner by using small molecules that target p53. (c)2010 AACR.

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.

Kaempferol, a phytoestrogen, suppressed triclosan-induced epithelial-mesenchymal transition and metastatic-related behaviors of MCF-7 breast cancer cells.

Science.gov (United States)

Lee, Geum-A; Choi, Kyung-Chul; Hwang, Kyung-A

2017-01-01

As a phytoestrogen, kaempferol is known to play a chemopreventive role inhibiting carcinogenesis and cancer progression. In this study, the influences of triclosan, an anti-bacterial agent recently known for an endocrine disrupting chemical (EDC), and kaempferol on breast cancer progression were examined by measuring their effects on epithelial-mesenchymal transition (EMT) and metastatic-related behaviors of MCF-7 breast cancer cells. Morphological changes of MCF-7 cells were observed, and a wound-healing assay was performed after the treatment of triclosan and kaempferol. The effects of triclosan and kaempferol on protein expression of EMT-related markers such as E-cadherin, N-cadherin, Snail, and Slug and metastasis-related markers such as cathepsin B, D, MMP-2 and -9 were investigated by Western blot assay. In microscopic observations, triclosan (10 -6 M) or E2 (10 -9 M) induced transition to mesenchymal phenotype of MCF-7 cells compared with the control. Co-treatment of ICI 182,780 (10 -8 M), an ER antagonist, or kaempferol (25μM) with E2 or triclosan restored the cellular morphology to an epithelial phenotype. In a wound-healing scratch and a transwell migration assay, triclosan enhanced migration and invasion of MCF-7 cells, but co-treatment of kaempferol or ICI 182,780 reduced the migration and invasion ability of MCF-7 cells to the control level. In addition, kaempferol effectively suppressed E2 or triclosan-induced protein expressions of EMT and metastasis promoting markers. Taken together, triclosan may be a distinct xenoestrogenic EDC to promote EMT, migration, and invasion of MCF-7 breast cancer cells through ER. On the other hand, kaempferol can be an alternative chemopreventive agent to effectively suppress the metastatic behavior of breast cancer induced by an endogenous estrogen as well as exogenous xenoestrogenic compounds including triclosan. Copyright © 2016 Elsevier B.V. All rights reserved.

E.coli O157:H7

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Treor Francis Fernandez

2008-06-01

Full Text Available The serious nature of the symptoms of haemorrhagic colitis and HUS and the apparent low infectious dose (<100 cells of E.coli O157:H7 places this food borne pathogen a most serious of known food borne pathogens. Persuasive evidence suggests that healthy cattle are a reservoir of O157 and they can enter the food chain to provide a source of exposure for humans. A possible route of transmission of O157 VTEC may involve infections initially in calves that shed their organism into faecal slurry that may be used on grazing grass. This provides potential for infection of other animals from which the organism may contaminate milk or carcasses at slaughter. Possible sources of VTEC in healthy animals other E.coli O157:H7 than cattle and a wider range of foodstuffs require further investigation. Many features of E.coli O157:H7 strains remain poorly understood. It includes: (i Role of virulent genes in the animal, (ii Mechanism of evolution of the organism, (iii The progress of individual cases of E.coli O157:H7 infection, and (iv The difference in incidence of infection in different geographical areas. [Veterinary World 2008; 1(3.000: 83-87

Dominant Role of HPV16 E7 in Anal Carcinogenesis

Science.gov (United States)

Thomas, Marie K.; Pitot, Henry C.; Liem, Amy; Lambert, Paul F.

2011-01-01

Ninety percent of anal cancer is associated with human papilloma viruses (HPVs). Using our previously established HPV transgenic mouse model for anal cancer, we tested the role of the individual oncogenes E6 and E7. K14E6 and K14E7 transgenic mice were treated with dimethylbenz[a]anthracene (DMBA) to the anal canal and compared to matched nontransgenic and doubly transgenic K14E6/E7 mice. K14E7 and K14E6/E7 transgenic mice developed anal tumors (papillomas, atypias and carcinomas combined) at significantly higher rates (88% and 100%, respectively) than either K14E6 or NTG mice (18% and 19%, respectively). Likewise, K14E7 and K14E6/E7 transgenic mice developed frank cancer (carcinomas) at significantly higher rates (85% and 85%, respectively) than either K14E6 or NTG mice (18% and 10%, respectively). These findings indicate that E7 is the more potent oncogene in anal cancer caused by HPVs. PMID:21999991

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...

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

International Nuclear Information System (INIS)

Islam, Shamima; Hassan, Ferdaus; Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Koide, Naoki; Naiki, Yoshikazu; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi

2007-01-01

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-α antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL). TNF-α might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-κB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed

7 CFR 15e.140 - Employment.

Science.gov (United States)

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Employment. 15e.140 Section 15e.140 Agriculture Office... OR ACTIVITIES CONDUCTED BY THE UNITED STATES DEPARTMENT OF AGRICULTURE § 15e.140 Employment. No... employment under any program or activity conducted by the agency. The definitions, requirements, and...

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

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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.

Modified Vero cell induced by Bifidobacterium bifidum inhibits enterohemorrhagic Escherichia coli O157:H7 cytopathic effect

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Tahamtan, Y.

2014-11-01

Full Text Available Enterohemorrhagic Escherichia coli (EHEC, such as E. coli O157:H7, are emerging food-borne pathogens worldwide. This micro-organism can damage the epithelial tissue of the large intestine. The cytotoxic effects can be neutralized by probiotics such as Bifidobacterium bifidum. Probiotics are viable cells that have beneficial effects on the health of the host. The preventing activity of B. bifidum against E. coli O157 was studied using a Vero cell model. Vero cell was pretreated with viable B. bifidum and incubated for either 3 h to 24 h and then collected from the cell to make modified Vero cell (MVC. Indirect antibacterial effects of B. bifidum were demonstrated by reduction of attachment of E. coli O157:H7 to MVC. The maximum reduction was resulted in pretreatment of Vero cell with B. bifidum for 24 h before infection. B. bifidum attenuated E. coli O157:H7 attachment to MVC up to 10 days of incubation. To our knowledge, MCV prevented Vero cell line injury induced by E. coli O157:H7. Therefore, B. bifidum can be used for inhibition of E. coli O157:H7 cytopathic effect (CPE in Vero cell model, even as pretreatment of the cell line.

Effect of RNase E deficiency on translocon protein synthesis in an RNase E-inducible strain of enterohemorrhagic Escherichia coli O157:H7.

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Lodato, Patricia B; Thuraisamy, Thujitha; Richards, Jamie; Belasco, Joel G

2017-07-06

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effect of 7-nitroindazole on body temperature and methamphetamine-induced dopamine toxicity.

Science.gov (United States)

Callahan, B T; Ricaurte, G A

1998-08-24

The present study was undertaken to examine the role of temperature on the ability of 7-nitroindazole (7-NI) to prevent methamphetamine-induced dopamine (DA) neurotoxicity. Male Swiss-Webster mice received methamphetamine alone or in combination with 7-NI at either room temperature (20+/-1 degrees C) or at 28+/-1 degrees C. At 20+/-1 degrees C, 7-NI produced hypothermic effects and afforded total protection against methamphetamine-induced DA depletions in the striatum. At 28+/-1 degrees C, 7-NI produced minimal effects on body temperature and failed to prevent methamphetamine-induced DA reductions. These findings indicate that the neuroprotection afforded by 7-NI is likely related to its ability to produce hypothermia because agents that produce hypothermia and/or prevent hyperthermia are known to attenuate methamphetamine-induced neurotoxicity.

The Subcellular Localisation of the Human Papillomavirus (HPV 16 E7 Protein in Cervical Cancer Cells and Its Perturbation by RNA Aptamers

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Özlem Cesur

2015-06-01

Full Text Available Human papillomavirus (HPV is the most common viral infection of the reproductive tract, affecting both men and women. High-risk oncogenic types are responsible for almost 90% of anogenital and oropharyngeal cancers including cervical cancer. Some of the HPV “early” genes, particularly E6 and E7, are known to act as oncogenes that promote tumour growth and malignant transformation. Most notably, HPV-16 E7 interacts with the tumour suppressor protein pRb, promoting its degradation, leading to cell cycle dysregulation in infected cells. We have previously shown that an RNA aptamer (termed A2 selectively binds to HPV16 E7 and is able to induce apoptosis in HPV16-transformed cervical carcinoma cell lines (SiHa through reduction of E7 levels. In this study, we investigated the effects of the A2 aptamer on E7 localisation in order to define its effects on E7 activity. We demonstrate for the first time that E7 localised to the plasma membrane. In addition, we show that A2 enhanced E7 localisation in the ER and that the A2-mediated reduction of E7 was not associated with proteasomal degradation. These data suggest that A2 perturbs normal E7 trafficking through promoting E7 ER retention.

Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

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Yongke Lu

2015-10-01

Full Text Available Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT, CYP2E1 knockout (KO or CYP2E1 humanized transgenic knockin (KI, mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA, an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These

Endothelial Semaphorin 7A promotes inflammation in seawater aspiration-induced acute lung injury.

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Zhang, Minlong; Wang, Li; Dong, Mingqing; Li, Zhichao; Jin, Faguang

2014-10-28

Inflammation is involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have shown that Semaphorin 7A (SEMA7A) promotes inflammation, there are limited reports regarding immunological function of SEMA7A in seawater aspiration-induced ALI. Therefore, we investigated the role of SEMA7A during seawater aspiration-induced ALI. Male Sprague-Dawley rats were underwent seawater instillation. Then, lung samples were collected at an indicated time for analysis. In addition, rat pulmonary microvascular endothelial cells (RPMVECs) were cultured and then stimulated with 25% seawater for indicated time point. After these treatments, cells samples were collected for analysis. In vivo, seawater instillation induced lung histopathologic changes, pro-inflammation cytokines release and increased expression of SEMA7A. In vitro, seawater stimulation led to pro-inflammation cytokine release, cytoskeleton remodeling and increased monolayer permeability in pulmonary microvascular endothelial cells. In addition, knockdown of hypoxia-inducible factor (HIF)-1α inhibited the seawater induced increase expression of SEMA7A. Meanwhile, knockdown of SEMA7A by specific siRNA inhibited the seawater induced aberrant inflammation, endothelial cytoskeleton remodeling and endothelial permeability. These results suggest that SEMA7A is critical in the development of lung inflammation and pulmonary edema in seawater aspiration-induced ALI, and may be a therapeutic target for this disease.

Cell death and impairment of glucose-stimulated insulin secretion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the β-cell line INS-1E

International Nuclear Information System (INIS)

Piaggi, Simona; Novelli, Michela; Martino, Luisa; Masini, Matilde; Raggi, Chiara; Orciuolo, Enrico; Masiello, Pellegrino; Casini, Alessandro; De Tata, Vincenzo

2007-01-01

The aim of this research was to characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the insulin-secreting β-cell line INS-1E. A sharp decline of cell survival (below 20%) was observed after 1 h exposure to TCDD concentrations between 12.5 and 25 nM. Ultrastructurally, β-cell death was characterized by extensive degranulation, appearance of autophagic vacuoles, and peripheral nuclear condensation. Cytotoxic concentrations of TCDD rapidly induced a dose-dependent increase in intracellular calcium concentration. Blocking calcium entry by EGTA significantly decreased TCDD cytotoxicity. TCDD was also able to rapidly induce mitochondrial depolarization. Interestingly, 1 h exposition of INS-1E cells to very low TCDD concentrations (0.05-1 nM) dramatically impaired glucose-stimulated but not KCl-stimulated insulin secretion. In conclusion, our results clearly show that TCDD exerts a direct β-cell cytotoxic effect at concentrations of 15-25 nM, but also markedly impairs glucose-stimulated insulin secretion at concentrations 20 times lower than these. On the basis of this latter observation we suggest that pancreatic β-cells could be considered a specific and sensitive target for dioxin toxicity

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

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Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

2017-06-01

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Repression of MHC class I transcription by HPV16E7 through interaction with a putative RXRβ motif and NF-κB cytoplasmic sequestration

International Nuclear Information System (INIS)

Li, Hui; Zhan, TaiLan; Li, Chang; Liu, Mugen; Wang, Qing K.

2009-01-01

Down-regulation of transcription of the MHC class I genes in HPV16 tumorigenic cells is partly due to HPV16E7 associated with the MHC class I promoter and repressed chromatin activation. In this study, we further demonstrated that HPV16E7 is physically associated with a putative RXRβ binding motif (GGTCA) of the proximal promoter of the MHC class I genes by using reporter transcriptional assays and chromatin immunoprecipitation assays. Our data also provide evidence that HPV16E7 inhibits TNF-α-induced up-regulation of MHC class I transcription by impaired nuclear translocation of NF-κB. More importantly, CaSki tumor cells treated with TSA and transfected with the constitutively active mutant form of IKK-α (which can activate NF-κB directly) showed a maximal level of up-regulation of MHC-I expression. Taken together, our results suggest that HPV16E7 may employ two independent mechanisms to ensure that either the constitutive or inducible transcription of MHC class I genes is down-regulated.

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...

Eleutheroside E inhibits doxorubicin-induced inflammation and ...

African Journals Online (AJOL)

Purpose: To identify the effects of eleutheroside E (EE) on apoptosis and inflammation induced by doxorubicin (DOX) in H9c2 cells and to investigate the underlying mechanisms. Methods: The effect of EE on H9c2 cell viability was determined using Cell Counting Kit-8 (CCK8). EE effect on DOX-induced apoptosis and ...

A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

Science.gov (United States)

Gagnon, David; Archambault, Jacques

2015-01-01

A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

Human Papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

Science.gov (United States)

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

2012-01-01

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone. PMID:22284893

Hippocampal infusions of apolipoprotein E peptides induce long-lasting cognitive impairment.

Science.gov (United States)

Eddins, Donnie; Klein, Rebecca C; Yakel, Jerrel L; Levin, Edward D

2009-04-29

The inheritance of the varepsilon4 allele of apolipoprotein E (ApoE4) and cholinergic system dysfunction have long been associated with the pathology of Alzheimer's disease (AD). Recently, in vitro studies have established a direct link between ApoE and cholinergic function in that synthetic peptides containing segments of the ApoE protein (ApoE(133-149) and ApoE(141-148)) interact with alpha7 nicotinic acetylcholine receptors (nAChRs) in the hippocampus. This raises the possibility that ApoE peptides may contribute to cognitive impairment in AD in that the hippocampus plays a key role in cognitive functioning. To test this, we acutely infused ApoE peptides into the ventral hippocampus of female Sprague-Dawley rats and assessed the resultant effects on radial-arm maze choice accuracy over a period of weeks after the infusion. Local ventral hippocampal infusion of ApoE peptides caused significant cognitive impairment in radial-arm maze learning that persisted several weeks after the acute infusion. This persisting deficit may be an important model for understanding the relationship between ApoE protein-induced neurotoxicity and cognitive impairment as well as serve as a platform for the development of new therapies to avoid neurotoxicity and cognitive decline.

Human Papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 Override the Normal Phosphoregulation of E6AP Enzymatic Activity.

Science.gov (United States)

Thatte, Jayashree; Banks, Lawrence

2017-11-15

The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution. IMPORTANCE Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a

Interferon β induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

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Chort, Alice; Alves, Sandro; Marinello, Martina; Dufresnois, Béatrice; Dornbierer, Jean-Gabriel; Tesson, Christelle; Latouche, Morwena; Baker, Darren P; Barkats, Martine; El Hachimi, Khalid H; Ruberg, Merle; Janer, Alexandre; Stevanin, Giovanni; Brice, Alexis; Sittler, Annie

2013-06-01

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Modulation of antigen presenting cell functions during chronic HPV infection

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Abate Assefa Bashaw

2017-12-01

Full Text Available High-risk human papillomaviruses (HR-HPV infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity.

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

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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.

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

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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.

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

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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.

Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7.

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Coleman, Leon G; Zou, Jian; Crews, Fulton T

2017-01-25

Toll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration. TLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles. We report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs. Our results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

Enhanced synthesis and secretion of apolipoprotein E from sciatic nerves of streptozotocin-induced diabetic rats after injury

International Nuclear Information System (INIS)

Ishibashi, S.; Yamada, N.; Oka, Y.

1988-01-01

To elucidate the pathogenesis of diabetic neuropathy, synthesis and secretion of apolipoprotein E (apo E) from sciatic nerves after injury was studied in normal and streptozotocin-induced diabetic rats. Seven, 14, 28, 45 and 59 days after making crush injury on sciatic nerves with concomitant administration of streptozotocin (50 mg/kg body weight), the nerves were taken out and incubated with [ 35 S]methionine. The [ 35 S]labeled apo E was precipitated with specific antiserum. The amounts of apo E secreted into medium by nerves of diabetic rats were 7 times greater than those of non-diabetic rats 7 days after injury. This enhanced secretion of apo E was relatively selective for this protein, since the ratio of the immunoprecipitable apo E to the TCA preciptitable protein in the medium increased in diabetic rats. Intriguing possibility deduced from these results is that the secretion of apo E is involved in the development of diabetic neuropathy

Indirubin Derivative 7-Bromoindirubin-3-Oxime (7Bio Attenuates Aβ Oligomer-Induced Cognitive Impairments in Mice

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Liping Chen

2017-11-01

Full Text Available Indirubins are natural occurring alkaloids extracted from indigo dye-containing plants. Indirubins could inhibit various kinases, and might be used to treat chronic myelocytic leukemia, cancer and neurodegenerative disorders. 7-bromoindirubin-3-oxime (7Bio, an indirubin derivative derived from indirubin-3-oxime, possesses inhibitory effects against cyclin-dependent kinase-5 (CDK5 and glycogen synthase kinase-3β (GSK3β, two pharmacological targets of Alzheimer's disease (AD. In this study, we have discovered that 2.3–23.3 μg/kg 7Bio effectively prevented β-amyloid (Aβ oligomer-induced impairments of spatial cognition and recognition without affecting bodyweight and motor functions in mice. Moreover, 7Bio potently inhibited Aβ oligomer-induced expression of interleukin-6 (IL-6 and tumor necrosis factor-α (TNF-α. Furthermore, 7Bio significantly prevented the decreased expression of synapsin-1 and PSD-95, biomarkers of pre-synaptic and post-synaptic proteins in Aβ oligomer-treated mice. The mean optical density (OD with hyper-phosphorylated tau (pTau, glial fibrillary acidic protein (GFAP and CD45 positive staining in the hippocampus of 7Bio-treated mice were significantly decreased compared to those of Aβ oligomer-treated mice. In addition, Western blotting analysis showed that 7Bio attenuated Aβ oligomer-decreased expression of pSer9-GSK3β. Those results suggested that 7Bio could potently inhibit Aβ oligomer-induced neuroinflammation, synaptic impairments, tau hyper-phosphorylation, and activation of astrocytes and microglia, which may contribute to the neuroprotective effects of 7Bio. Based on these findings, we expected that 7Bio might be developed as a novel anti-AD lead compound.

Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E

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Lorena M. C. Cursino

2016-02-01

Full Text Available Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4’-trimethoxy-4-prenylstilbene (1, 4-methoxyderricidine (2, lonchocarpine (3, 4-hydroxylonchocarpine (4, 4-methoxylonchocarpine (5, 5-hydroxy-4’,7-dimethoxy-6-prenylflavanone (6, 4’-hydroxyisolonchocarpine (7, 4’-methoxyisolonchocarpine (8, 3’,4’,7-trimethoxyflavone (9, 3’,4’-methylenedioxy-7-methoxyflavone (10, and 2,2-dimethyl-chromone-5,4’-hydroxy-5’-methoxyflavone (11. Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK and the eukaryotic elongation factor 2 (eEF2. The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.

Deformation of conjugate compliant fault zones induced by the 2013 Mw7.7 Baluchistan (Pakistan) earthquake

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Dutta, Rishabh; Wang, Teng; Feng, Guangcai; Harrington, Jonathan; Vasyura-Bathke, Hannes; Jónsson, Sigurjón

2017-04-01

Strain localizations in compliant fault zones (with elastic moduli lower than the surrounding rocks) induced by nearby earthquakes have been detected using geodetic observations in a few cases in the past. Here we observe small-scale changes in interferometric Synthetic Aperture Radar (InSAR) measurements along multiple conjugate faults near the rupture of the 2013 Mw7.7 Baluchistan (Pakistan) earthquake. After removing the main coseismic deformation signal in the interferograms and correcting them for topography-related phase, we observe 2-3 cm signal along several conjugate faults that are 15-30 km from the mainshock fault rupture. These conjugate compliant faults have strikes of N30°E and N45°W. The sense of motion indicates left-lateral deformation across the N30°E faults and right-lateral deformation across the N45°W faults, which suggests the conjugate faults were subjected to extensional coseismic stresses along the WSW-ENE direction. The spacing between the different sets of faults is around 5 to 8 km. We explain the observed strain localizations as an elastic response of the compliant conjugate faults induced by the Baluchistan earthquake. Using 3D Finite Element models (FEM), we impose coseismic static displacements due to the earthquake along the boundaries of the FEM domain to reproduce the coseismic stress changes acting across the compliant faults. The InSAR measurements are used to constrain the geometry and rigidity variations of the compliant faults with respect to the surrounding rocks. The best fitting models show the compliant fault zones to have a width of 0.5 km to 2 km and a reduction of the shear modulus by a factor of 3 to 4. Our study yields similar values as were found for compliant fault zones near the 1992 Landers and the 1999 Hector Mine earthquakes in California, although here the strain localization is occurring on more complex conjugate sets of faults.

Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

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Cao, Bo; Chai, Chunxiang; Zhao, Sishun

2015-12-01

Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in Escherichia coli O157:H7▿

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Burt, Sara A.; van der Zee, Ruurd; Koets, Ad P.; de Graaff, Anko M.; van Knapen, Frans; Gaastra, Wim; Haagsman, Henk P.; Veldhuizen, Edwin J. A.

2007-01-01

The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7. PMID:17526792

E6 and E7 Gene Polymorphisms in Human Papillomavirus Types-58 and 33 Identified in Southwest China.

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Zuyi Chen

Full Text Available Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV. The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216 and HPV-58 (E6, E7: n = 405 E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0. The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8 software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N were observed in both HPV-33 and HPV-58 E6.

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.

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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.

Design of a colicin E7 based chimeric zinc-finger nuclease

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Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

2014-08-01

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

Novel spin-electronic properties of BC7 sheets induced by strain

International Nuclear Information System (INIS)

Xu, Lei; Dai, ZhenHong; Sui, PengFei; Sun, YuMing; Wang, WeiTian

2014-01-01

Based on first-principles calculations, the authors have investigated the electronic and magnetic properties of BC 7 sheets with different planar strains. It is found that metal–semiconductor transition appears at the biaxial strain of 15.5%, and the sheets are characteristic of spin-polarized semiconductor with a zero band-gap. The band-gap rapidly increases with strain, and reaches a maximum value of 0.60 eV at the strain of 20%. Subsequently, the band-gap decreases until the strain reaches up to 22% and shows a semiconductor-half metal transformation. It will further present metal properties until the strain is up to the maximum value of 35%. The magnetic moments also have some changes induced by biaxial strain. The numerical analysis shows that the two-dimensional distortions have great influences on the magnetic moments. The novel spin-electronic properties make BC 7 sheets have potential applications in future spintronic nanodevices

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

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Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

2010-03-01

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

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Lee, Kiwon; Lee, Ah-Young; Kwon, Yunhee Kim; Kwon, Hyockman

2011-01-01

Highlights: → Integration of HPV into host genome critical for activation of E6 and E7 oncogenes. → BAF53 is essential for higher-order chromatin structure. → BAF53 knockdown suppresses E6 and E7 from HPV integrants, but not from episomal HPVs. → BAF53 knockdown decreases H3K9Ac and H4K12Ac on P105 promoter of integrated HPV 18. → BAF53 knockdown restores the p53-dependent signaling pathway in HeLa and SiHa cells. -- Abstract: Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor

Fbw7α and Fbw7γ Collaborate To Shuttle Cyclin E1 into the Nucleolus for Multiubiquitylation

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Bhaskaran, Nimesh; van Drogen, Frank; Ng, Hwee-Fang; Kumar, Raman; Ekholm-Reed, Susanna; Peter, Matthias

2013-01-01

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G1-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G1 and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCFFbw7 (also known as SCFCdc4), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCFFbw7α in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCFFbw7γ. Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCFFbw7γ prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation. PMID:23109421

Angiotensin-(1-7) attenuates hyposmolarity-induced ANP secretion via the Na+-K+ pump.

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Shah, Amin; Oh, Young-Bin; Shan, Gao; Song, Chang Ho; Park, Byung-Hyun; Kim, Suhn Hee

2010-09-01

The alteration in osmolarity challenges cell volume regulation, a vital element for cell survival. Hyposmolarity causes an increase in cell volume. Recently, it has been reported that the renin-angiotensin system (RAS) plays a role in cell volume regulation. We investigated the effect of angiotensin-(1-7) [Ang-(1-7)] on hyposmolarity-induced atrial natriuretic peptide (ANP) secretion in normal and diabetic (DM) rat atria and modulation of the effect of Ang-(1-7) by the Na(+)-K(+) pump. Using isolated control rat atria, we observed that perfusion of hyposmotic solution into the atria increased ANP secretion. When Ang-(1-7) [0.1 microM or 1 microM] was perfused in a hyposmolar solution, it decreased the hyposmolarity-induced ANP secretion in a dose-dependent manner. This effect of Ang-(1-7) could be mediated by the Na(+)-K(+) pump, since ouabain, an Na(+)-K(+) pump inhibitor, significantly decreased the effect of Ang-(1-7) on hyposmolarity-induced ANP secretion. In contrast, N(omega) Nitro-l-arginine methyl ester hydrochloride (l-NAME) did not modify the effect of Ang-(1-7) on the hyposmolarity-induced ANP secretion. Interestingly, the ANP secretion was increased robustly by the perfusion of the hyposmolar solution in the DM atria, as compared to the control atria. However, the inhibitory effect of Ang-(1-7) on the hyposmolarity-induced ANP secretion was not observed in the DM atria. In the DM atria, atrial contractility was significantly increased. Taken together, we concluded that Ang-(1-7) attenuated hyposmolarity-induced ANP secretion via the Na(+)-K(+) pump and a lack of Ang-(1-7) response in DM atria may partly relate to change in Na(+)-K(+) pump activity. Copyright 2010 Elsevier Inc. All rights reserved.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation

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Di Bonito P

2017-06-01

Full Text Available Paola Di Bonito,1 Chiara Chiozzini,2 Claudia Arenaccio,2 Simona Anticoli,2 Francesco Manfredi,2 Eleonora Olivetta,2 Flavia Ferrantelli,2 Emiliana Falcone,3 Anna Ruggieri,3 Maurizio Federico2 1Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy; 2National AIDS Center, Istituto Superiore di Sanità, Rome, Italy; 3Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy Abstract: We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPV E7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8+ T-cell immune response against both Nef and E7. Conversely, no CD8+ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA

Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

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Garzón-Ospina, Diego; Forero-Rodríguez, Johanna; Patarroyo, Manuel A

2014-12-13

The msp-7 gene has become differentially expanded in the Plasmodium genus; Plasmodium vivax has the highest copy number of this gene, several of which encode antigenic proteins in merozoites. DNA sequences from thirty-six Colombian clinical isolates from P. vivax (pv) msp-7E, -7F and -7L genes were analysed for characterizing and studying the genetic diversity of these pvmsp-7 members which are expressed during the intra-erythrocyte stage; natural selection signals producing the variation pattern so observed were evaluated. The pvmsp-7E gene was highly polymorphic compared to pvmsp-7F and pvmsp-7L which were seen to have limited genetic diversity; pvmsp-7E polymorphism was seen to have been maintained by different types of positive selection. Even though these copies seemed to be species-specific duplications, a search in the Plasmodium cynomolgi genome (P. vivax sister taxon) showed that both species shared the whole msp-7 repertoire. This led to exploring the long-term effect of natural selection by comparing the orthologous sequences which led to finding signatures for lineage-specific positive selection. The results confirmed that the P. vivax msp-7 family has a heterogeneous genetic diversity pattern; some members are highly conserved whilst others are highly diverse. The results suggested that the 3'-end of these genes encode MSP-7 proteins' functional region whilst the central region of pvmsp-7E has evolved rapidly. The lineage-specific positive selection signals found suggested that mutations occurring in msp-7s genes during host switch may have succeeded in adapting the ancestral P. vivax parasite population to humans.

Recombinant Listeria vaccines containing PEST sequences are potent immune adjuvants for the tumor-associated antigen human papillomavirus-16 E7.

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Sewell, Duane A; Shahabi, Vafa; Gunn, George R; Pan, Zhen-Kun; Dominiecki, Mary E; Paterson, Yvonne

2004-12-15

Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH(2) terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8(+) T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8(+) T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8(+) T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.

Autotaxin is induced by TSA through HDAC3 and HDAC7 inhibition and antagonizes the TSA-induced cell apoptosis.

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Li, Song; Wang, Baolu; Xu, Yan; Zhang, Junjie

2011-02-12

Autotaxin (ATX) is a secreted glycoprotein with the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive lysophospholipid involved in diverse biological actions. ATX is highly expressed in some cancer cells and contributes to their tumorigenesis, invasion, and metastases, while in other cancer cells ATX is silenced or expressed at low level. The mechanism of ATX expression regulation in cancer cells remains largely unknown. In the present study, we demonstrated that trichostatin A (TSA), a well-known HDAC inhibitor (HDACi), significantly induced ATX expression in SW480 and several other cancer cells with low or undetectable endogenous ATX expression. ATX induction could be observed when HDAC3 and HDAC7 were down-regulated by their siRNAs. It was found that HDAC7 expression levels were low in the cancer cells with high endogenous ATX expression. Exogenous over-expression of HDAC7 inhibited ATX expression in these cells in a HDAC3-dependent manner. These data indicate that HDAC3 and HDAC7 collaboratively suppress ATX expression in cancer cells, and suggest that TSA induce ATX expression by inhibiting HDAC3 and HDAC7. The biological significance of this regulation mechanism was revealed by demonstrating that TSA-induced ATX protected cancer cells against TSA-induced apoptosis by producing LPA through its lysoPLD activity, which could be reversed by BrP-LPA and S32826, the inhibitors of the ATX-LPA axis. We have demonstrated that ATX expression is repressed by HDAC3 and HDAC7 in cancer cells. During TSA treatment, ATX is induced due to the HDAC3 and HDAC7 inhibition and functionally antagonizes the TSA-induced apoptosis. These results reveal an internal HDACi-resistant mechanism in cancer cells, and suggest that the inhibition of ATX-LPA axis would be helpful to improve the efficacy of HDACi-based therapeutics against cancer.

Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

International Nuclear Information System (INIS)

Fu, Wen; Gorodeski, George I; McCormick, Tom; Qi, Xiaoping; Luo, Liping; Zhou, Lingyin; Li, Xin; Wang, Bing-Cheng; Gibbons, Heidi E; Abdul-Karim, Fadi W

2009-01-01

The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X 7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X 7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X 7 action. Skin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X 7 -null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X 7 pore formation) were determined by confocal laser microscopy. (a) Co-application on the skin of the P2X 7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X 7 -receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X 7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the

Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells

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Owa C

2013-09-01

Full Text Available Chie Owa, Michael E Messina Jr, Reginald HalabyDepartment of Biology, Montclair State University, Montclair, NJ, USABackground: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3.Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells.Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability.Conclusion: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.Keywords: triptolide, MCF-7 breast cancer cells, apoptosis, lysosomes, lysosomal membrane permeabilization (LMP

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

ERK/CANP rapid signaling mediates 17β-estradiol-induced proliferation of human breast cancer cell line MCF-7 cells.

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Wang, Guo-Sheng; Huang, Yan-Gang; Li, Huan; Bi, Shi-Jie; Zhao, Jin-Long

2014-01-01

17β-estradiol (E2) exerts its functions through both genomic and non-genomic signaling pathways. Because E2 is important in breast cancer development, we investigated whether its actions in promoting breast cancer cell proliferation occur through the non-genomic signaling pathway via extracellular signal-regulated kinase 1/2 (ERK1/2)/calcium-activated neutral protease (CANP). MCF-7 breast cancer cells were treated with ERKl/2 inhibitor (PD98059) or CANP inhibitor (calpeptin) before exposure to 1×10(-8) M E2. MTT colorimetry and flow cytometry were used to analyze effects on cell proliferation and cell cycle progression, respectively. Expression of phosphorylated-ERK (p-ERK), total ERK, and Capn4 proteins were assessed by Western blotting. Cell proliferation increased in cells treated with E2 for 24 h (P<0.05), and the proportion of cells in G0/G1 was decreased, accompanied by accelerated G1/S. Calpeptin pre-treatment significantly inhibited the E2-induced proliferation of MCF-7 cells (P<0.05), while also ameliorating the effects of E2 on cell cycle progression. Further, expression of p-ERK was rapidly up-regulated (after 10 min) by E2 (P<0.05), an effect that persisted 16 h after E2 exposure but which was significantly inhibited by PD98059 (P<0.05). Finally, expression of Capn4 protein was rapidly up-regulated in E2-exposed cells (P<0.05), but this change was significantly inhibited by PD98059 or calpeptin (P<0.05) pre-treatment. Thus, the rapid, non-genomic ERK/CANP signaling pathway mediates E2-induced proliferation of human breast cancer cells.

Sarcosine induces increase in HER2/neu expression in androgen-dependent prostate cancer cells

DEFF Research Database (Denmark)

Dahl, Malin; Bouchelouche, Pierre; Kramer-Marek, Gabriela

2011-01-01

Increasing evidence suggests that Human epidermal growth factor receptor 2 (HER2/neu) is involved in progression of prostate cancer. Recently, sarcosine was reported to be highly increased during prostate cancer progression, and exogenous sarcosine induces an invasive phenotype in benign prostate...... epithelial cells. The aim of this work was to investigate the effect of sarcosine on HER2/neu expression in prostate cancer cell lines LNCaP (androgen dependent), PC-3 and DU145 (both androgen independent). Relative amounts of HER2/neu and androgen receptor (AR) transcripts were determined using RT...... that sarcosine is involved in the regulation of the oncoprotein HER2/neu. Thus, sarcosine may induce prostate cancer progression by increased HER2/neu expression. However, detailed information on cellular mechanisms remains to be elucidated....

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

International Nuclear Information System (INIS)

Jing, Xu; Ren, Dongmei; Wei, Xinbing; Shi, Huanying; Zhang, Xiumei; Perez, Ruth G.; Lou, Haiyan; Lou, Hongxiang

2013-01-01

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke. - Highlights: • E7G activates Nrf2 in astrocytes. • E7G stimulates expression of Nrf2-mediated cytoprotective proteins in astrocytes. • E7G protects astrocytes against OGD-induced cell death and apoptosis. • The neuroprotective effect of E7G involves the Nrf2/ARE pathway. • E7G protects rats against cerebral ischemic injury

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

Energy Technology Data Exchange (ETDEWEB)

Jing, Xu [Department of Pharmacology, School of Medicine, Shandong University, Jinan 250012 (China); Ren, Dongmei [Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, Shandong University, Jinan 250012 (China); Wei, Xinbing; Shi, Huanying; Zhang, Xiumei [Department of Pharmacology, School of Medicine, Shandong University, Jinan 250012 (China); Perez, Ruth G. [Health Science Center, Paul L. Foster School of Medicine, Texas Tech University, El Paso, TX, 79905 (United States); Lou, Haiyan, E-mail: louhaiyan@sdu.edu.cn [Department of Pharmacology, School of Medicine, Shandong University, Jinan 250012 (China); Lou, Hongxiang [Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, Shandong University, Jinan 250012 (China)

2013-12-15

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke. - Highlights: • E7G activates Nrf2 in astrocytes. • E7G stimulates expression of Nrf2-mediated cytoprotective proteins in astrocytes. • E7G protects astrocytes against OGD-induced cell death and apoptosis. • The neuroprotective effect of E7G involves the Nrf2/ARE pathway. • E7G protects rats against cerebral ischemic injury.

Genetic responses induced in olive roots upon colonization by the biocontrol endophytic bacterium Pseudomonas fluorescens PICF7.

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Elisabetta Schilirò

Full Text Available Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1 were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

SagE induces highly effective protective immunity against Streptococcus iniae mainly through an immunogenic domain in the extracellular region.

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Sun, Yun; Sun, Li; Xing, Ming-qing; Liu, Chun-sheng; Hu, Yong-hua

2013-11-12

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen of a wide range of farmed fish. S. iniae possesses a virulence-associated streptolysin S cluster composed of several components, one of which is SagE. SagE a transmembrane protein with one major extracellular region named ECR. This study aimed to develop a SagE-based DNA candidate vaccine against streptococcosis and examine the immunoprotective mechanism of the vaccine. We constructed a DNA vaccine, pSagE, based on the sagE gene and examined its immunological property in a Japanese flounder (Paralichthys olivaceus) model. The results showed that at 7 days post-vaccination, expression of SagE at transcription and translation levels was detected in the tissues of the vaccinated fish. After challenge with S. iniae at one and two months post-vaccination, pSagE-vaccinated fish exhibited relative percent survival (RPS) of 95% and 88% respectively. Immunological analysis showed that (i) pSagE significantly upregulated the expression of a wide range of immune genes, (ii) pSagE induced the production of specific serum antibodies that bound whole-cell S. iniae, and (iii) treatment of S. iniae with pSagE-induced antibodies blocked bacterial invasion of host cells. To localize the immunoprotective domain of SagE, the ECR-expressing DNA vaccine pSagEECR was constructed. Immunization analysis showed that flounder vaccinated with pSagEECR exhibited a RPS of 68%, and that pSagEECR induced serum antibody production and immune gene expression in a manner similar to, though to lower magnitudes than, those induced by pSagE. We in this study developed a DNA vaccine, pSagE, which induces highly protective immunity against S. iniae. The protective effect of pSagE is probably due to its ability to elicit systemic immune response, in particular that of the humoral branch, which leads to production of specific serum antibodies that impair bacterial infection. These results add insights to the immunoprotective mechanism

Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

International Nuclear Information System (INIS)

Barrow-Laing, Lisa; Chen Wei; Roman, Ann

2010-01-01

The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.

Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle

International Nuclear Information System (INIS)

Lee, Choongho; Wooldridge, Tonia R.; Laimins, Laimonis A.

2007-01-01

The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another α-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span

E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection.

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Yueting Zheng

2016-01-01

Full Text Available Interferons (IFNs are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC and TERT-immortalized normal human diploid fibroblasts (HDF-TERT. IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib, a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

Vitamins C and E for asthma and exercise-induced bronchoconstriction.

Science.gov (United States)

Wilkinson, Mark; Hart, Anna; Milan, Stephen J; Sugumar, Karnam

2014-06-17

The association between dietary antioxidants and asthma or exercise-induced bronchoconstriction (EIB) is not fully understood. Vitamin C and vitamin E are natural antioxidants that are predominantly present in fruits and vegetables; inadequate vitamin E intake is associated with airway inflammation. It has been postulated that the combination may be more beneficial than either single antioxidant for people with asthma and exercise-induced bronchoconstriction. To assess the effects of supplementation of vitamins C and E versus placebo (or no vitamin C and E supplementation) on exacerbations and health-related quality of life (HRQL) in adults and children with chronic asthma. To also examine the potential effects of vitamins C and E on exercise-induced bronchoconstriction in people with asthma and in people without a diagnosis of asthma who experience symptoms only on exercise. Trials were identified from the Cochrane Airways Review Group Specialised Register and from trial registry websites. Searches were conducted in September 2013. We included randomised controlled trials of adults and children with a diagnosis of asthma. We separately considered trials in which participants had received a diagnosis of exercise-induced bronchoconstriction (or exercise-induced asthma). Trials comparing vitamin C and E supplementation versus placebo were included. We included trials in which asthma management for treatment and control groups included similar background therapy. Short-term use of vitamins C and E at the time of exacerbation or for cold symptoms in people with asthma is outside the scope of this review. Two review authors independently screened the titles and abstracts of potential studies and subsequently screened full-text study reports for inclusion. We used standard methods as expected by The Cochrane Collaboration. It was not possible to aggregate the five included studies (214 participants). Four studies (206 participants) addressed the question of whether

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

International Nuclear Information System (INIS)

Chandrasekharan, Sabarinath; Kandasamy, Krishna Kumar; Dayalan, Pavithra; Ramamurthy, Viraragavan

2013-01-01

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

Energy Technology Data Exchange (ETDEWEB)

Chandrasekharan, Sabarinath, E-mail: csab@bio.psgtech.ac.in [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India); Kandasamy, Krishna Kumar [Max Planck Institute for Biology of Ageing, Cologne (Germany); Dayalan, Pavithra; Ramamurthy, Viraragavan [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India)

2013-08-02

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

Understanding the role of the catalase/peroxide genes in H2O2 resistance of E. coli serotype O157:H7 biofilms

Science.gov (United States)

Introduction: Escherichia coli serotype O157:H7 defenses against H2O2 include the peroxiredoxin AhpC and three catalases: KatG (catalase-peroxidase), KatE (catalase), and the plasmid-encoded KatP (catalase/peroxidase). AhpC, KatG, and KatP are induced by OxyR in exponential phase, while KatE is indu...

Novel spin-electronic properties of BC{sub 7} sheets induced by strain

Energy Technology Data Exchange (ETDEWEB)

Xu, Lei; Dai, ZhenHong, E-mail: zhdai@ytu.edu.cn; Sui, PengFei; Sun, YuMing; Wang, WeiTian [Computational Physics Laboratory, Institute of Opto-Electronic Information Science and Technology, Yantai University, Yantai 264005 (China)

2014-11-01

Based on first-principles calculations, the authors have investigated the electronic and magnetic properties of BC{sub 7} sheets with different planar strains. It is found that metal–semiconductor transition appears at the biaxial strain of 15.5%, and the sheets are characteristic of spin-polarized semiconductor with a zero band-gap. The band-gap rapidly increases with strain, and reaches a maximum value of 0.60 eV at the strain of 20%. Subsequently, the band-gap decreases until the strain reaches up to 22% and shows a semiconductor-half metal transformation. It will further present metal properties until the strain is up to the maximum value of 35%. The magnetic moments also have some changes induced by biaxial strain. The numerical analysis shows that the two-dimensional distortions have great influences on the magnetic moments. The novel spin-electronic properties make BC{sub 7} sheets have potential applications in future spintronic nanodevices.

A novel strategy to improve antigen presentation for active immunotherapy in cancer. Fusion of the human papillomavirus type 16 E7 antigen to a cell penetrating peptide

International Nuclear Information System (INIS)

Granadillo, Milaid; Torrens, Isis; Guerra, Maribel

2012-01-01

Facilitating the delivery of exogenous antigens to antigen-presenting cells, ensuing processing and presentation via the major histocompatibility complex class I and induction of an effective immune response are fundamental for an effective therapeutic cancer vaccine. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel cell-penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus anti-lipopolysaccharide factor protein (LALF 32-51 ) linked to human papillomavirus 16 E7 antigen (LALF 32-51 -E7). In this work, we demonstrated that the immunization with LALF 32-51 -E7 using the TC-1 mouse model induces a potent and long-lasting anti-tumor response supported on an effective E7-specific CD8 +T -cell response. The finding that therapeutic immunization with LALF 32-51 or E7 alone, or an admixture of LALF32-51 and E7, does not induce significant tumor reduction indicates that covalent linkage between LALF 32-51 and E7 is required for the anti-tumor effect. These results support the use of this novel cell-penetrating peptide as an efficient means for delivering therapeutic targets into cellular compartments with the induction of a cytotoxic CD8 +T lymphocyte immune response. This approach is promissory for the treatment of tumors associated with the human papillomavirus 16, which is responsible for the 50% of cervical cancer cases worldwide and other malignancies. Furthermore, protein-based vaccines can circumvent the major histocompatibility complex specificity limitation associated with peptide vaccines providing a greater extent in their application

Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

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Fu Wen

2009-04-01

Full Text Available Abstract Background The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7 action. Methods Skin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA. Changes in cytosolic calcium or in ethidium bromide influx (P2X7 pore formation were determined by confocal laser microscopy. Results (a Co-application on the skin of the P2X7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28 the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d Levels of P2X7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for

Protocadherin-7 induces bone metastasis of breast cancer

Energy Technology Data Exchange (ETDEWEB)

Li, Ai-Min [Department of Orthopedics, The 5th Central Hospital of Tianjin, Tianjin (China); Tian, Ai-Xian [Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Zhang, Rui-Xue [Department of Clinical Laboratory Diagnosis, Tianjin Medical University, Tianjin (China); Ge, Jie [Department of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Sun, Xuan [Department of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Cao, Xu-Chen, E-mail: caoxuch@126.com [Department of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China)

2013-07-05

Highlights: •PCDH7 is overexpression in high bone metastatic MDA-MB-231 cells. •PCDH7 is up-regulation in bone metastatic breast cancer tissues. •Suppression of PCDH7 inhibits cell proliferation, migration, and invasion in vitro. •PCDH7 induces breast cancer bone metastasis in vivo. -- Abstract: Breast cancer had a propensity to metastasize to bone, resulting in serious skeletal complications associated with poor outcome. Previous study showed that Protocadherin-7 (PCDH7) play an important role in brain metastatic breast cancer, however, the role of PCDH7 in bone metastatic breast cancer has never been explored. In the present study, we found that PCDH7 expression was up-regulation in bone metastatic breast cancer tissues by real-time PCR and immunohistochemistry assays. Furthermore, suppression of PCDH7 inhibits breast cancer cell proliferation, migration, and invasion in vitro by MTT, scratch, and transwell assays. Most importantly, overexpression of PCDH7 promotes breast cancer cell proliferation and invasion in vitro, and formation of bone metastasis in vivo. These data provide an important insight into the role of PCDH7 in bone metastasis of breast cancer.

Protocadherin-7 induces bone metastasis of breast cancer

International Nuclear Information System (INIS)

Li, Ai-Min; Tian, Ai-Xian; Zhang, Rui-Xue; Ge, Jie; Sun, Xuan; Cao, Xu-Chen

2013-01-01

Highlights: •PCDH7 is overexpression in high bone metastatic MDA-MB-231 cells. •PCDH7 is up-regulation in bone metastatic breast cancer tissues. •Suppression of PCDH7 inhibits cell proliferation, migration, and invasion in vitro. •PCDH7 induces breast cancer bone metastasis in vivo. -- Abstract: Breast cancer had a propensity to metastasize to bone, resulting in serious skeletal complications associated with poor outcome. Previous study showed that Protocadherin-7 (PCDH7) play an important role in brain metastatic breast cancer, however, the role of PCDH7 in bone metastatic breast cancer has never been explored. In the present study, we found that PCDH7 expression was up-regulation in bone metastatic breast cancer tissues by real-time PCR and immunohistochemistry assays. Furthermore, suppression of PCDH7 inhibits breast cancer cell proliferation, migration, and invasion in vitro by MTT, scratch, and transwell assays. Most importantly, overexpression of PCDH7 promotes breast cancer cell proliferation and invasion in vitro, and formation of bone metastasis in vivo. These data provide an important insight into the role of PCDH7 in bone metastasis of breast cancer

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ.

Science.gov (United States)

Di Matteo, A; Franceschini, M; Paiardini, A; Grottesi, A; Chiarella, S; Rocchio, S; Di Natale, C; Marasco, D; Vitagliano, L; Travaglini-Allocatelli, C; Federici, L

2017-09-18

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

2-Azidoalkoxy-7-hydro-8-oxoadenine derivatives as TLR7 agonists inducing dendritic cell maturation.

Science.gov (United States)

Weterings, Jimmy J; Khan, Selina; van der Heden van Noort, Gerbrand J; Melief, Cornelis J M; Overkleeft, Herman S; van der Burg, Sjoerd H; Ossendorp, Ferry; van der Marel, Gijsbert A; Filippov, Dmitri V

2009-04-15

The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.

Cimetidine attenuates vinorelbine-induced phlebitis in mice by militating E-selectin expression.

Science.gov (United States)

Wang, Zhuo; Ma, Lijuan; Wang, Xuebin; Cai, Heping; Huang, Jin; Liu, Jiyong; Hu, Jinhong; Su, Dingfeng

2014-08-01

We investigated E-selectin expression in mice and rabbits with vinorelbine-induced phlebitis and the effect of cimetidine. To find the relationship between E-selectin expression and vinorelbine-induced phlebitis. Mouse and rabbit model of vinorelbine-induced phlebitis was established by intravenous infusion of vinorelbine. Pathological observation, molecular-biological determination of E-selectin and protein function of it was evaluated. Grossly, we observed swelling, edema and cord-like vessel changes in mice receiving vinorelbine but only mild edema in mice pretreated with cimetidine. Pathological scoring yielded a total score of 37 for vinorelbine-treated mice and 17 for mice pretreated with cimetidine (P phlebitis in mice probably by suppressing increased expression of E-selectin.

Selenium and vitamin E inhibit radiogenic and chemically induced transformation in vitro via different mechanisms

International Nuclear Information System (INIS)

Borek, C.; Ong, A.; Mason, H.; Donahue, L.; Biaglow, J.E.

1986-01-01

Results from in vivo and in vitro studies showing that antioxidants may act as anticarcinogens support the role of active oxygen in carcinogenesis and provide impetus for exploring the functions of dietary antioxidants in cancer prevention by using in vitro models. The authors examined the single and combined effects of selenium, a component of glutathione peroxidase, and vitamin E, a known antioxidant, on cell transformation induced in C3H/10T-1/2 cells by x-rays, benzo[a]pyrene, or tryptophan pyrolysate and on the levels of cellular scavenging systems peroxide destruction. Incubation of C3H/10T-1/2 cells with 2.5 μM Na 2 SeO 3 (selenium) or with 7 μM α-tocopherol succinate (vitamin E) 24 hr prior to exposure to x-rays or the chemical carcinogens resulted in an inhibition of transformation by each of the antioxidants with an additive-inhibitory action when the two nutrients were combined. Cellular pretreatment with selenium resulted in increased levels of cellular glutathione peroxidase, catalase, and nonprotein thiols (glutathione) and in an enhanced destruction of peroxide. The results support our earlier studies showing that free radical-mediated events play a role in radiation and chemically induced transformation. They indicate that selenium and vitamin E act alone and in additive fashion as radioprotecting and chemopreventing agents. The results further suggest that selenium confers protection in part by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown while vitamin E appears to confer its protection by and alternate complementary mechanism

In-beam PET measurement of $^{7}Li^{3+}$ irradiation induced $\\beta^+}$-activity

CERN Document Server

Priegnitz, M; Parodi, K; Sommerer, F; Fiedler, F; Enghardt, W

2008-01-01

At present positron emission tomography (PET) is the only feasible method of an in situ and non-invasive monitoring of patient irradiation with ions. At the experimental carbon ion treatment facility of the Gesellschaft für Schwerionenforschung (GSI) Darmstadt an in-beam PET scanner has been integrated into the treatment site and lead to a considerable quality improvement of the therapy. Since ions other than carbon are expected to come into operation in future patient treatment facilities, it is highly desirable to extend in-beam PET also to other therapeutic relevant ions, e.g. 7Li. Therefore, by means of the in-beam PET scanner at GSI the β+-activity induced by 7Li3+ ions has been investigated for the first time. Targets of PMMA, water, graphite and polyethylene were irradiated with monoenergetic, pencil-like beams of 7Li3+ with energies between 129.1 A MeV and 205.3 A MeV and intensities ranging from 3.0 × 107 to 1.9 × 108 ions s−1. This paper presents the measured β+-activity profiles as well as d...

32 CFR 147.7 - Guideline E-Personal conduct.

Science.gov (United States)

2010-07-01

... questionnaire, personal history statement, or similar form used to conduct investigations, determine employment... 32 National Defense 1 2010-07-01 2010-07-01 false Guideline E-Personal conduct. 147.7 Section 147... Adjudication § 147.7 Guideline E—Personal conduct. (a) The concern. Conduct involving questionable judgment...

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli

International Nuclear Information System (INIS)

Crosby, R.M.; Richardson, K.K.; Craft, T.R.; Benforado, K.B.; Liber, H.L.; Skopek, T.R.

1988-01-01

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT - human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli., DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism

Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.

Science.gov (United States)

Baumschlager, Armin; Aoki, Stephanie K; Khammash, Mustafa

2017-11-17

Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.

Suberoyl bis-hydroxamic acid induces p53-dependent apoptosis of MCF-7 breast cancer cells

Institute of Scientific and Technical Information of China (English)

Zhi-gang ZHUANG; Fei FEI; Ying CHEN; Wei JIN

2008-01-01

Aim: To study the effects of suberoyl bis-hydroxamic acid (SBHA), an inhibitor of histone deacetylases, on the apoptosis of MCF-7 breast cancer cells. Meth-ods: Apoptosis in MCF-7 cells induced by SBHA was demonstrated by flow cytometric analysis, morphological observation, and DNA ladder. Mitochondrial membrane potential (△ψm) was measured using the fluorescent probe JC-1. The expressions of p53, p21, Bax, and PUMA were determined using RT-PCR or Western blotting analysis after the MCF-7 cells were treated with SBHA or p53 siRNA. Results: SBHA induced apoptosis in MCF-7 cells. The expressions of p53, p21, Bax, and PUMA were induced, and △ψm collapsed after treatment with SBHA. p53 siRNA abrogated the SBHA-induced apoptosis and the expressions of p53, p21, Bax, and PUMA. Conclusion: The activation of the p53 pathway is involved in SBHA-induced apoptosis in MCF-7 cells.

Red Mold Rice Mitigates Oral Carcinogenesis in 7,12-Dimethyl-1,2-Benz[a]anthracene-Induced Oral Carcinogenesis in Hamster

Directory of Open Access Journals (Sweden)

Ruei-Lan Tsai

2011-01-01

Full Text Available The prevalence of oral tumor has exponentially increased in recent years; however, the effective therapies or prevention strategies are not sufficient. Red mold rice is a traditional Chinese food, and several reports have demonstrated that red mold rice had an anti-tumor effect. However, the possible anti-tumor mechanisms of the red mold rice are unclear. In this study, we examined the anti-tumor effect of red mold rice on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA-induced oral tumor in hamster. The ethanol extract of red mold rice (RMRE treatment significantly decreases the levels of DMBA-induced reactive oxygen species, nitro oxide and prostaglandin E2 than those of the lovastatin-treated group (P < .001. Moreover, RMRE decreases the formation of oral tumor induced by DMBA. Monacolin K, monascin, ankaflavin or other red mold rice metabolites had been reported to decrease inflammation and oxidative stress and exerted anti-tumor effects. Therefore, we evaluated the anti-inflammation and anti-oxidative stress effects of monacolin K, monascin, ankaflavin and citrinin in lipopolysaccharide-treated RAW264.7 cells. We found that RMRE reduced the LPS-induced nitrite levels in RAW264.7 cells better than monacolin K, monascin, ankaflavin or citrinin (P < .05.

Role of IGFBP7 in Diabetic Nephropathy: TGF-β1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells.

Directory of Open Access Journals (Sweden)

Jun Watanabe

Full Text Available Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN, and TGF-β1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs. Urine samples from Japanese patients with type 2 diabetes (n = 46 were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-β1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-β1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-β1-induced epithelial to mesenchymal transition (EMT. In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037, eGFR (r = -0.376, p = 0.01, urinary β2-microglobulin (r = 0.385, p = 0.008, and urinary N-acetyl-beta-D-glucosaminidase (NAG (r = 0.502, p = 0.000. A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-β1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-β1-induced tubular injury in DN.

The Curcumin Analogue 1,5-Bis(2-hydroxyphenyl-1,4-pentadiene-3-one Induces Apoptosis and Downregulates E6 and E7 Oncogene Expression in HPV16 and HPV18-Infected Cervical Cancer Cells

Directory of Open Access Journals (Sweden)

Felicia Paulraj

2015-06-01

Full Text Available In an effort to study curcumin analogues as an alternative to improve the therapeutic efficacy of curcumin, we screened the cytotoxic potential of four diarylpentanoids using the HeLa and CaSki cervical cancer cell lines. Determination of their EC50 values indicated relatively higher potency of 1,5-bis(2-hydroxyphenyl-1,4-pentadiene-3-one (MS17, 1.03 ± 0.5 μM; 2.6 ± 0.9 μM and 1,5-bis(4-hydroxy-3-methoxyphenyl-1,4-pentadiene-3-one (MS13, 2.8 ± 0.4; 6.7 ± 2.4 μM in CaSki and HeLa, respectively, with significantly greater growth inhibition at 48 and 72 h of treatment compared to the other analogues or curcumin. Based on cytotoxic and anti-proliferative activity, MS17 was selected for comprehensive apoptotic studies. At 24 h of treatment, fluorescence microscopy detected that MS17-exposed cells exhibited significant morphological changes consistent with apoptosis, corroborated by an increase in nucleosomal enrichment due to DNA fragmentation in HeLa and CaSki cells and activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR also detected significant down-regulation of HPV18- and HPV16-associated E6 and E7 oncogene expression following treatment. The overall data suggests that MS17 treatment has cytotoxic, anti-proliferative and apoptosis-inducing potential in HPV-positive cervical cancer cells. Furthermore, its role in down-regulation of HPV-associated oncogenes responsible for cancer progression merits further investigation into its chemotherapeutic role for cervical cancer.

Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T-cell-Mediated Tumor Control in the Genital Tract.

Science.gov (United States)

Sun, Yun-Yan; Peng, Shiwen; Han, Liping; Qiu, Jin; Song, Liwen; Tsai, Yachea; Yang, Benjamin; Roden, Richard B S; Trimble, Cornelia L; Hung, Chien-Fu; Wu, T-C

2016-02-01

Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16(+) cervical cancer (TC-1 luc). We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8(+) T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8(+) T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8(+) T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8(+) T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Our results support future clinical translation using cervicovaginal TA-HPV vaccination. ©2015 American Association for Cancer Research.

Local HPV Recombinant Vaccinia Boost Following Priming with an HPV DNA Vaccine Enhances Local HPV-Specific CD8+ T Cell Mediated Tumor Control in the Genital Tract

Science.gov (United States)

Sun, Yun-Yan; Peng, Shiwen; Han, Liping; Qiu, Jin; Song, Liwen; Tsai, Yachea; Yang, Benjamin; Roden, Richard B.S.; Trimble, Cornelia L.; Hung, Chien-Fu; Wu, T-C

2015-01-01

Purpose Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)-infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Experimental Design Here we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than IM delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16+ cervical cancer (TC-1 luc). Results We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8+ T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8+ T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8+ T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8+ T cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Conclusions Our results support future clinical translation using cervicovaginal TA-HPV vaccination. PMID:26420854

Topical W-7 inhibits ultraviolet radiation-induced melanogenesis in Skh:HR2 pigmented hairless mice

International Nuclear Information System (INIS)

Dowdy, J.C.; Anthony, F.A.; Costlow, M.E.

1995-01-01

We studied the effect of N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) on ultraviolet radiation (UVR)-induced melanogenesis (tanning) in Skh:HR2 pigmented hairless mice. Topically pretreated mice were exposed to subminimal edematogenic as well as edematogenic UVR doses to establish whether W-7-UVR-induced edema prophylaxis allows increased melanogenesis while preventing edema. Ultraviolet light-irradiated vehicle control animals developed visible trans; however, both W-7-treated groups failed to tan. Topical W-7 before UVR exposure inhibited UVR induction of dopa oxidase activity in melanocytes by 49% (P=0.029) and inhibited UVR-induced deposition of melanin in the epidermis by 88% (P=0.006). Topical W-7 blocked 23% of the UVR but this blockage could not account for the inhibition of dopa oxidase and melanization. We conclude that, in addition to preventing edema, W-7 inhibits UVR-induced melanogenesis, possibly by affecting Ca 2+ -calmodulin and/or protein kinase C-dependent processes. (au) 30 refs

Topical W-7 inhibits ultraviolet radiation-induced melanogenesis in Skh:HR2 pigmented hairless mice

Energy Technology Data Exchange (ETDEWEB)

Dowdy, J.C. [Univ. of Memphis, Div. of Molecular Sciences and Microbiology, Memphis, Tennessee (United States); Anthony, F.A.; Costlow, M.E. [Schering-Plough HealthCare Products, Inc., Advanced Product Research, Memphis, Tennessee (United States)

1995-08-01

We studied the effect of N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) on ultraviolet radiation (UVR)-induced melanogenesis (tanning) in Skh:HR2 pigmented hairless mice. Topically pretreated mice were exposed to subminimal edematogenic as well as edematogenic UVR doses to establish whether W-7-UVR-induced edema prophylaxis allows increased melanogenesis while preventing edema. Ultraviolet light-irradiated vehicle control animals developed visible trans; however, both W-7-treated groups failed to tan. Topical W-7 before UVR exposure inhibited UVR induction of dopa oxidase activity in melanocytes by 49% (P=0.029) and inhibited UVR-induced deposition of melanin in the epidermis by 88% (P=0.006). Topical W-7 blocked 23% of the UVR but this blockage could not account for the inhibition of dopa oxidase and melanization. We conclude that, in addition to preventing edema, W-7 inhibits UVR-induced melanogenesis, possibly by affecting Ca{sup 2+}-calmodulin and/or protein kinase C-dependent processes. (au) 30 refs.

E. S. R. study of free radicals formed in the irradiated DNA-Ro 7-0582 complex

Energy Technology Data Exchange (ETDEWEB)

Washino, K; Kuwabara, M; Yoshii, G [Hokkaido Univ., Sapporo (Japan)

1979-01-01

The effect of Ro 7-0582 (1-(2-hydroxy-3-methoxypropyl)-2-nitro-imidazole) on the formation of free radicals in ..gamma..-irradiated dry DNA has been investigated. Dry samples of DNA-Ro 7-0582 and DNA nucleotide-Ro 7-0582 were prepared, and e.s.r. spectra observed at 77 K immediately after gamma-irradiation. The samples were then warmed to 297 K for 30 min, and the spectra again observed at 77 K. The sensitizer brought about an increase of 30 to 40% in radical formation in DNA. The results indicated that Ro 7-0582 acts as an efficient electron scavenger on the TMP and dAMP moieties, increasing the incidence of sugar damage. Since TMP and dAMP form a complementary pair in the DNA double helix, the increase in double strand breaks induced by electron-affinic compounds seems to be responsible for the molecular mechanism of radiosensitization in living cells.

Loss of FBXW7 and accumulation of MCL1 and PLK1 promote paclitaxel resistance in breast cancer.

Science.gov (United States)

Gasca, Jessica; Flores, Maria Luz; Giráldez, Servando; Ruiz-Borrego, Manuel; Tortolero, María; Romero, Francisco; Japón, Miguel A; Sáez, Carmen

2016-08-16

FBXW7 is a component of SCF (complex of SKP1, CUL1 and F-box-protein)-type ubiquitin ligases that targets several oncoproteins for ubiquitination and degradation by the proteasome. FBXW7 regulates cellular apoptosis by targeting MCL1 for ubiquitination. Recently, we identified PLK1 as a new substrate of FBXW7 modulating the intra-S-phase DNA-damage checkpoint. Taxanes are frequently used in breast cancer treatments, but the acquisition of resistance makes these treatments ineffective. We investigated the role of FBXW7 and their substrates MCL1 and PLK1 in regulating the apoptotic response to paclitaxel treatment in breast cancer cells and their expression in breast cancer tissues. Paclitaxel-sensitive MDA-MB-468 and a paclitaxel-resistant MDA-MB-468R subclone were used to study the role of FBXW7 and substrates in paclitaxel-induced apoptosis. Forced expression of FBXW7 or downregulation of MCL1 or PLK1 restored sensitivity to paclitaxel in MDA-MB-468R cells. By contrary, FBXW7-silenced MDA-MB-468 cells became resistant to paclitaxel. The expression of FBXW7 and substrates were studied in 296 invasive carcinomas by immunohistochemistry and disease-free survival was analyzed in a subset of patients treated with paclitaxel. In breast cancer tissues, loss of FBXW7 correlated with adverse prognosis markers and loss of FBXW7 and MCL1 or PLK1 accumulation were associated with diminished disease-free survival in paclitaxel-treated patients. We conclude that FBXW7 regulates the response to paclitaxel by targeting MCL1 and PLK1 in breast cancer cells and thus targeting these substrates may be a valuable adjunct for paclitaxel treatment. Also, FBXW7, MCL1 and PLK1 may be relevant predictive markers of tumor progression and response to paclitaxel treatment.

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....

Anti-Inflammatory Activity of Heterocarpin from the Salt Marsh Plant Corydalis heterocarpa in LPS-Induced RAW 264.7 Macrophage Cells

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You Ah Kim

2015-08-01

Full Text Available The inhibitory effect of three chromones 1–3 and two coumarins 4–5 on the production of nitric oxide (NO was evaluated in LPS-induced RAW 264.7 macrophage cells. Among the compounds tested heterocarpin (1, a furochromone, significantly inhibited its production in a dose-dependent manner. In addition, heterocarpin suppressed prostaglandin E2 (PGE2 production and expression of cytokines such as inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (COX-2, tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β and interleukin-6 (IL-6.

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

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Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

2004-07-16

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

Vitamin E for antipsychotic-induced tardive dyskinesia.

Science.gov (United States)

Soares-Weiser, Karla; Maayan, Nicola; Bergman, Hanna

2018-01-17

Antipsychotic (neuroleptic) medication is used extensively to treat people with chronic mental illnesses. Its use, however, is associated with adverse effects, including movement disorders such as tardive dyskinesia (TD) - a problem often seen as repetitive involuntary movements around the mouth and face. Vitamin E has been proposed as a treatment to prevent or decrease TD. The primary objective was to determine the clinical effects of vitamin E in people with schizophrenia or other chronic mental illness who had developed antipsychotic-induced TD.The secondary objectives were:1. to examine whether the effect of vitamin E was maintained as duration of follow-up increased;2. to test the hypothesis that the use of vitamin E is most effective for those with early onset TD (less than five years) SEARCH METHODS: We searched the Cochrane Schizophrenia Group Trials Register (July 2015 and April 2017), inspected references of all identified studies for further trials and contacted authors of trials for additional information. We included reports if they were controlled trials dealing with people with antipsychotic-induced TD and schizophrenia who remained on their antipsychotic medication and had been randomly allocated to either vitamin E or to a placebo, no intervention, or any other intervention. We independently extracted data from these trials and we estimated risk ratios (RR) or mean differences (MD), with 95% confidence intervals (CI). We assumed that people who left early had no improvement. We assessed risk of bias and created a 'Summary of findings' table using GRADE. The review now includes 13 poorly reported randomised trials (total 478 people), all participants were adults with chronic psychiatric disorders, mostly schizophrenia, and antipsychotic-induced TD. There was no clear difference between vitamin E and placebo for the outcome of TD: not improved to a clinically important extent (6 RCTs, N = 264, RR 0.95, 95% CI 0.89 to 1.01, low-quality evidence

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.

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.

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

Science.gov (United States)

Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Cytoprotective Effects of E-α-(4-Methoxyphenyl-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC--A Novel and Non-Cytotoxic HO-1 Inducer.

Directory of Open Access Journals (Sweden)

Kai B Kaufmann

Full Text Available Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1, is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7

Identifying Conformational-Selection and Induced-Fit Aspects in the Binding-Induced Folding of PMI from Markov State Modeling of Atomistic Simulations.

Science.gov (United States)

Paul, Fabian; Noé, Frank; Weikl, Thomas R

2018-03-27

Unstructured proteins and peptides typically fold during binding to ligand proteins. A challenging problem is to identify the mechanism and kinetics of these binding-induced folding processes in experiments and atomistic simulations. In this Article, we present a detailed picture for the folding of the inhibitor peptide PMI into a helix during binding to the oncoprotein fragment 25-109 Mdm2 obtained from atomistic, explicit-water simulations and Markov state modeling. We find that binding-induced folding of PMI is highly parallel and can occur along a multitude of pathways. Some pathways are induced-fit-like with binding occurring prior to PMI helix formation, while other pathways are conformational-selection-like with binding after helix formation. On the majority of pathways, however, binding is intricately coupled to folding, without clear temporal ordering. A central feature of these pathways is PMI motion on the Mdm2 surface, along the binding groove of Mdm2 or over the rim of this groove. The native binding groove of Mdm2 thus appears as an asymmetric funnel for PMI binding. Overall, binding-induced folding of PMI does not fit into the classical picture of induced fit or conformational selection that implies a clear temporal ordering of binding and folding events. We argue that this holds in general for binding-induced folding processes because binding and folding events in these processes likely occur on similar time scales and do exhibit the time-scale separation required for temporal ordering.

Intestinal CYP2E1: A mediator of alcohol-induced gut leakiness

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Christopher B. Forsyth

2014-01-01

Full Text Available Chronic alcohol use can result in many pathological effects including alcoholic liver disease (ALD. While alcohol is necessary for the development of ALD, only 20–30% of alcoholics develop alcoholic steatohepatitis (ASH with progressive liver disease leading to cirrhosis and liver failure (ALD. This suggests that while chronic alcohol consumption is necessary it is not sufficient to induce clinically relevant liver damage in the absence of a secondary risk factor. Studies in rodent models and alcoholic patients show that increased intestinal permeability to microbial products like endotoxin play a critical role in promoting liver inflammation in ALD pathogenesis. Therefore identifying mechanisms of alcohol-induced intestinal permeability is important in identifying mechanisms of ALD and for designing new avenues for therapy. Cyp2e1 is a cytochrome P450 enzyme that metabolizes alcohol has been shown to be upregulated by chronic alcohol use and to be a major source of oxidative stress and liver injury in alcoholics and in animal and in vitro models of chronic alcohol use. Because Cyp2e1 is also expressed in the intestine and is upregulated by chronic alcohol use, we hypothesized it could play a role in alcohol-induced intestinal hyperpermeability. Our in vitro studies with intestinal Caco-2 cells and in mice fed alcohol showed that circadian clock proteins CLOCK and PER2 are required for alcohol-induced permeability. We also showed that alcohol increases Cyp2e1 protein and activity but not mRNA in Caco-2 cells and that an inhibitor of oxidative stress or siRNA knockdown of Cyp2e1 prevents the increase in CLOCK or PER2 proteins and prevents alcohol-induced hyperpermeability. With our collaborators we have also shown that Cyp2e1 knockout mice are resistant to alcohol-induced gut leakiness and liver inflammation. Taken together our data support a novel Cyp2e1-circadian clock protein mechanism for alcohol-induced gut leakiness that could provide new

The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

Science.gov (United States)

Khan, Omar M.; Carvalho, Joana; Spencer-Dene, Bradley; Mitter, Richard; Frith, David; Snijders, Ambrosius P.; Wood, Stephen A.

2018-01-01

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations. PMID:29346117

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

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity

Science.gov (United States)

Ermakova, Elena; Miller, Michelle C; Nesmelova, Irina V; López-Merino, Lara; Berbís, Manuel Alvaro; Nesmelov, Yuri; Tkachev, Yaroslav V; Lagartera, Laura; Daragan, Vladimir A; André, Sabine; Cañada, F Javier; Jiménez-Barbero, Jesús; Solís, Dolores; Gabius, Hans-Joachim; Mayo, Kevin H

2013-01-01

The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 103 M−1 and K2 = 3.4 ± 0.8 × 103 M−1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family. PMID:23376190

Laser-induced damage study of polymer PMMA; Motale-e-ye padid-e-ye khesarat-e mavvad-e polimeri PMMA dar moghabel-e barik-e-ye laizer

Energy Technology Data Exchange (ETDEWEB)

Mansour, N

2001-07-01

This article presents the results of bulk laser-induced damage measurements in polymer PMMA at 532 nm and 1064 nm for nanosecond laser pulses. The damage thresholds were measured for focused spot sizes ranging over two orders of magnitude. In this work, self-focusing effects were verified to be absent by measurements of breakdown thresholds using both linearly and circularly polarized light. At both 1064 nm and 532 nm, the dependence of the breakdown field, E{sub B}, on the spot size, {omega}, was empirically determined to be E{sub B} = C/{radical}{omega}, where C depends on the wavelength. The extracted value for C({lambda}) at 1064 nm is larger by a factor of 5 than at 532 nm. Possible reasons for this strong dispersion and mechanism for laser-induced damage in polymer materials will be discussed.

Effects of Flavin7 on allergen induced hyperreactivity of airways

Directory of Open Access Journals (Sweden)

Franova S

2009-12-01

Full Text Available Abstract Some studies have suggested that the polyphenolic compounds might reduce the occurrence of asthma symptoms. The aim of our experiments was to evaluate the effects of 21 days of the flavonoid Flavin7 administration on experimentally induced airway inflammation in ovalbumin-sensitized guinea pigs. We assessed tracheal smooth muscle reactivity by an in vitro muscle-strip method; changes in airway resistance by an in vivo plethysmographic method; histological picture of tracheal tissue; and the levels of interleukin 4 (IL-4, and interleukin 5 (IL-5 in bronchoalveolar lavage fluid (BALF. Histological investigation of tracheal tissue and the concentrations of the inflammatory cytokines IL-4 and IL-5 in BALF were used as indices of airway inflammation. Administration of Flavin7 caused a significant decrease of specific airway resistance after histamine nebulization and a decline in tracheal smooth muscle contraction amplitude in response to bronchoconstricting mediators. Flavin7 minimized the degree of inflammation estimated on the basis of eosinophil calculation and IL-4 and IL-5 concentrations. In conclusion, administration of Flavin7 showed bronchodilating and anti-inflammatory effects on allergen-induced airway inflammation.

Oenothera laciniata inhibits lipopolysaccharide induced production of nitric oxide, prostaglandin E2, and proinflammatory cytokines in RAW264.7 macrophages.

Science.gov (United States)

Yoon, Weon-Jong; Ham, Young Min; Yoo, Byoung-Sam; Moon, Ji-Young; Koh, Jaesook; Hyun, Chang-Gu

2009-04-01

We elucidated the pharmacological and biological effects of Oenothera laciniata extracts on the production of inflammatory mediators in macrophages. The CH(2)Cl(2) fraction of O. laciniata extract effectively inhibited LPS-induced NO, PGE(2), and proinflammatory cytokine production in RAW264.7 cells. These inhibitory effects of the CH(2)Cl(2) fraction of O. laciniata were accompanied by decreases in the expression of iNOS and COX-2 proteins and iNOS, COX-2, TNF-alpha, IL-1beta, and IL-6 mRNA. Asiatic acid and quercetin were present in the HPLC fingerprint of the O. laciniata extract. We tested the potential application of O. laciniata extract as a cosmetic material by performing primary skin irritation tests. In New Zealand white rabbits, primary irritation tests revealed that application of O. laciniata extracts (1%) did not induce erythema or edema formation. Human skin primary irritation tests were performed on the normal skin (upper back) of 30 volunteers to determine if any material in O. laciniata extracts had irritation or sensitization potential. In these assays, O. laciniata extracts did not induce any adverse reactions. Based on these results, we suggest that O. laciniata extracts be considered possible anti-inflammatory candidates for topical application.

Novel TRAIL sensitizer Taraxacum officinale F.H. Wigg enhances TRAIL-induced apoptosis in Huh7 cells.

Science.gov (United States)

Yoon, Ji-Yong; Cho, Hyun-Soo; Lee, Jeong-Ju; Lee, Hyo-Jung; Jun, Soo Young; Lee, Jae-Hye; Song, Hyuk-Hwan; Choi, SangHo; Saloura, Vassiliki; Park, Choon Gil; Kim, Cheol-Hee; Kim, Nam-Soon

2016-04-01

TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Pressure-induced magnetic collapse and metallization of TlF e1.6S e2

Science.gov (United States)

Naumov, P. G.; Filsinger, K.; Shylin, S. I.; Barkalov, O. I.; Ksenofontov, V.; Qi, Y.; Palasyuk, T.; Schnelle, W.; Medvedev, S. A.; Greenblatt, M.; Felser, C.

2017-08-01

The crystal structure, magnetic ordering, and electrical resistivity of TlF e1.6S e2 were studied at high pressures. Below ˜7 GPa , TlF e1.6S e2 is an antiferromagnetically ordered semiconductor with a ThC r2S i2 -type structure. The insulator-to-metal transformation observed at a pressure of ˜7 GPa is accompanied by a loss of magnetic ordering and an isostructural phase transition. In the pressure range ˜7.5 -11 GPa a remarkable downturn in resistivity, which resembles a superconducting transition, is observed below 15 K. We discuss this feature as the possible onset of superconductivity originating from a phase separation in a small fraction of the sample in the vicinity of the magnetic transition.

Measurement of the Induced Proton Polarization Pn in the 12C(e, e', p) reaction

International Nuclear Information System (INIS)

Woo, R.J.; Barkhuff, David; Bertozzi, William; Jian-ping Chen; Dan Dale; G. Dodson; K.A. Dow; Marty Epstein; Manouchehr Farkhondeh; Mike Finn; Shalev Gilad; Mark K. Jones; Kyungseon Joo; James Kelly; Stanley Kowalski; Bob Lourie; Richard Madey; Dimitri Margaziotis; Pete Markowitz; Justin McIntyre; Christoph Mertz; Brian Milbrath; Joseph Mitchell; Charles F. Perdrisat; Vina Punjabi; Paul Rutt; Adam Sarty; D. Tieger; C. Tschalaer; William Turchinetz; Paul E. Ulmer; S.P. Van Verst; C. Vellidis; Glen Warren; Lawrence Weinstein

1998-01-01

The first measurements of the induced proton polarization Pn for the 12C(e,e',p) reaction are reported. The experiment was performed at quasifree kinematics for energy and momentum transfer (w,q) = (294 MeV, 765 MeV/c) and sampled a missing momentum range of 0-250 MeV/c. The induced polarization arises from final-state interactions and for these kinematics is dominated by the real part of the spin-orbit optical potential. The distorted-wave impulse approximation provides good agreement with data for the 1 p3/2 shell. The data for the continuum suggest that both the 1s1/2 shell and underlying l > 1 configurations contribute

Hyperoxia-induced ciliary loss and oxidative damage in an in vitro bovine model: The protective role of antioxidant vitamins E and C

International Nuclear Information System (INIS)

Al-Shmgani, Hanady S.; Moate, Roy M.; Sneyd, J. Robert; Macnaughton, Peter D.; Moody, A. John

2012-01-01

Highlights: ► A new bovine bronchial model for studying hyperoxia-induced cilia loss is presented. ► Hyperoxia-induced cilia loss was associated with increased sloughing of cells. ► Hyperoxia led to higher epithelial glutathione levels, evidence of oxidative stress. ► Hyperoxia led to increased DNA damage (Comet), and lipid peroxidation (TBARS). ► Vitamins C and E partially protected against hyperoxia-induced cilia loss. -- Abstract: Although elevated oxygen fraction is used in intensive care units around the world, pathological changes in pulmonary tissue have been shown to occur with prolonged exposure to hyperoxia. In this work a bovine bronchus culture model has been successfully used to evaluate the effects of hyperoxia on ciliated epithelium in vitro. Samples were cultured using an air interface method and exposed to normoxia, 21% O 2 or hyperoxia, 95% O 2 . Cilial coverage was assessed using scanning electron microscopy (SEM). Tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione) were used to assess whether the hyperoxia caused significant oxidative stress. Hyperoxia caused a time-dependent decline (t ½ = 3.4 d compared to 37.1 d under normoxia) in cilial coverage (P 6 compared to 1.97 ± 0.23 × 10 6 ml −1 after 6 d), many apparently intact, in the medium (P −1 g −1 after 6 d; P −1 for hyperoxia and normoxia, respectively); % tail DNA (18.7 ± 2.2 versus 11.1 ± 1.5); protein carbonyls (P −1 versus 189 ± 15 μmol g −1 ). Vitamins E (10 −7 M) and C (10 −6 or 10 −7 M) alone or in combination (10 −7 M and 10 −6 M, respectively) had a significant protective effect on the hyperoxia-induced reduction in percentage cilial coverage (P < 0.05). In conclusion, hyperoxia caused damage to cultured bovine bronchial epithelium and denudation of cilia. The

A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

International Nuclear Information System (INIS)

Hickey, E.W.

1988-01-01

Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH 0 ) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH 0 induced behavior in E. coli and S. typhimurium by quantitation of H 2 35 SO 4 labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH 0 5.0 or shifted from growth at pH 0 7.0 to 5.0. Internal pH (pH i ) changes occurring during steady state growth at low pH 0 , and on shifting from pH 0 7.0 to 5.0, were measured using 14 C-benzoic acid uptake

Suppression of Antitumor Immune Responses by Human Papillomavirus through Epigenetic Downregulation of CXCL14

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Louis Cicchini

2016-05-01

Full Text Available High-risk human papillomaviruses (HPVs are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK, CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression.

Upregulation of HPV E6 and E7 genes in human cervix carcinoma cell lines leads to both an increase in radiation resistance and alterations in apoptosis

International Nuclear Information System (INIS)

Kamradt, M.; Krueger, E.; McCall, A.; Manker, C.; Rudnick, S.; Erlich, E.; Khodarev, N; Vaughan, A.T.M.

1996-01-01

Purpose: Stage I-III cervical cancer is commonly treated with external beam radiotherapy with curative intent. We have shown that the presence of the artificial steroid dexamethasone can increase the radiation resistance of HPV+ve cervical tumors. Dexamethasone treatment enhances cell proliferation by upregulation of HPV 18 E6 and E7 genes through a glucocorticoid responsive element within the HPV promoter region. The goal of this study is to understand the mechanism whereby steroid treatment produces enhanced radioresistance in cervical tumors. Methods: Four cervical tumor cell lines and one squamous carcinoma cell line (SQ-20B) were used. Each cell line was exposed to 0 - 8 Gy of gamma irradiation in the presence or absence of 1 μM dexamethasone (Dx) and assayed for cell survival using a clonogenic assay. Parallel samples were examined by fluorescence microscopy after Triton X-100 permeabilization and propidium iodide staining 1, 2 and 4 days post irradiation. Nuclei containing the characteristic multi-lobular structure and condensed appearance of apoptosis were recorded. To confirm the presence of apoptosis in these cells a terminal deoxynucleotide transferase staining reaction was carried out to label the free DNA ends characteristic of apoptosis. Results: Administration of Dx increased radiation resistance in all HPV+ve E6 and E7 expressing cell lines studied, a similar effect was seen in two additional HPV 18+ve cervical carcinoma cell lines ME180 and MS751 (data not shown). No effect was seen in the HPV-ve cell lines, or C41 K6 containing anti-sense E6 and E7 genes. Introduction of the anti-sense E6 and E7 sequences into C41 did not affect their ability to undergo radiation-induced apoptosis though Dx administration reduced the amount of apoptosis seen. Conversely, Dx treatment of the HeLa cell line increased radioresistance but did not affect the ability of these cells to engage apoptosis. Conclusion: All (5) cervical cell lines studied containing HPV 18

IL-7 splicing variant IL-7δ5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

International Nuclear Information System (INIS)

Pan, Deshun; Liu, Bing; Jin, Xiaobao; Zhu, Jiayong

2012-01-01

Highlights: ► This study confirms the role of IL-7δ5 in breast cancer cell proliferation. ► IL-7δ5 promotes breast cancer cell proliferation and cell cycle progression. ► IL-7δ5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7δ5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7δ5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27 kip1 expression. Mechanistically, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7δ5. In conclusion, our findings demonstrate that IL-7δ5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target for human breast cancer therapeutics intervention.

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.

Asociación entre la presencia de anticuerpos anti-Ras y anti-VPH16 E4/E7 y lesiones intraepiteliales del cérvix Association between anti-Ras and anti-HPV16 E4/E7 antibodies with cervical intraepithelial lesions

Directory of Open Access Journals (Sweden)

Sara Vázquez-Corzo

2003-10-01

Full Text Available OBJETIVO: Determinar si anticuerpos séricos contra E4, E7 y Ras pueden ser utilizados como marcadores de lesiones tempranas del cérvix uterino asociadas al virus del papiloma humano. MATERIAL Y MÉTODOS: Entre marzo de 1999 y abril de 2000 se realizó un estudio sero-epidemiológico de casos y controles en la clínica de displasias del Hospital General Doctor Gea González, en la Ciudad de México, en 116 muestras de suero para evaluar la presencia de anticuerpos anti-E4, E7 y Ras utilizando un ELISA de captura. Se estimaron razones de momios e intervalos de confianza de 95% RESULTADOS: Anticuerpos anti-E7 se asociaron a mujeres con lesiones NIC III, mientras que anticuerpos anti-E4 y anti-Ras fueron más frecuentes en lesiones NIC I-II. Al evaluar el perfil de anticuerpos que presentaron las mujeres, encontramos que a anticuerpos contra dos proteínas predicen la existencia de una lesión NIC I-II, y b la presencia de tres anticuerpos predicen una lesión NIC III. CONCLUSIONES: La detección de anticuerpos séricos contra E4, E7 y Ras en combinación con otras técnicas de diagnóstico, podrían ser de utilidad para detectar oportunamente a mujeres con lesiones tempranas asociadas al Virus del Papiloma Humano y en riesgo de desarrollar cáncer.OBJECTIVE: To evaluate whether serum antibodies anti-E4, E7 and Ras could be used as markers for early cervical lesions associated with HPV (human papillomavirus. MATERIAL AND METHODS: A seroepidemiological case-control study was conducted between March 1999 and April 2000 at the dysplasia clinic of Hospital General Doctor Gea Gonzalez, in Mexico City, to evaluate the presence of antibodies anti-E4, E7, and Ras through a sandwich ELISA. Analysis was done using odds ratios and 95% confidence intervals. RESULTS: Anti-E7 antibodies were associated to women with CIN III lesions, while anti-E4 and Ras antibodies were strongly associated with CIN I-II lesions. The antibody profile of women with different

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

Science.gov (United States)

Uyttendaele, M; Taverniers, I; Debevere, J

2001-05-21

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.

The field induced e31,f piezoelectric and Rayleigh response in barium strontium titanate thin films

International Nuclear Information System (INIS)

Garten, L. M.; Trolier-McKinstry, S.

2014-01-01

The electric field induced e 31,f piezoelectric response and tunability of Ba 0.7 Sr 0.3 TiO 3 (70:30) and Ba 0.6 Sr 0.4 TiO 3 (60:40) thin films on MgO and silicon was measured. The relative dielectric tunabilities for the 70:30 and 60:40 compositions on MgO were 83% and 70%, respectively, with a dielectric loss of less than 0.011 and 0.004 at 100 kHz. A linear increase in induced piezoelectricity to −3.0 C/m 2 and −1.5 C/m 2 at 110 kV/cm was observed in Ba 0.6 Sr 0.4 TiO 3 on MgO and Ba 0.7 Sr 0.3 TiO 3 on Si. Hysteresis in the piezoelectric and dielectric response of the 70:30 composition films was consistent with the positive irreversible dielectric Rayleigh coefficient. Both indicate a ferroelectric contribution to the piezoelectric and dielectric response over 40–80 °C above the global paraelectric transition temperature.

Protective Effect of Vitamin E on Nicotine Induced Reproductive ...

African Journals Online (AJOL)

Protective Effect of Vitamin E on Nicotine Induced Reproductive Toxicity in ... The health implications of cigarette smoking and ..... toxic byproducts of many metabolic processes in ... Male infertility, clinical ... rats: A possible role of cessation.

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.

Radiation-induced ηe-modes

International Nuclear Information System (INIS)

Shukla, P.K.; Yu, M.Y.

1990-01-01

Impurity radiation in a plasma can cause not only static instabilities, but also dynamic instabilities related to the drift and acoustic waves. Radiative instabilities are of much interest because they are associated with relatively high frequency and short wavelength fluctuations, which have been suspected to be responsible for anomalous electron energy transport in tokamak edge plasmas. In this paper, we consider radiation-induced η e instabilities, taking into account electrostatic effects as well as density and temperature inhomogeneities. Also included are the effects of finite gyroradius and dissipation. It is found that the latter can cause strong linear coupling between the modes of interest. The resulting instabilities can have larger growth rates than the static radiative instability. Analytical expressions for the growth rates and instability regimes are given for the limiting cases of practical interest. In particular, it is shown that the η e -mode can couple to both radiation and dissipation to cause resistive instabilities. The parameter regimes of the original radiative as well as the dissipative modes are thereby broadened and shifted because of the interaction. (author) 3 refs

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...

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

International Nuclear Information System (INIS)

Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun; Xiao, Shaobo

2010-01-01

Research highlights: → FMDV L pro inhibits poly(I:C)-induced IFN-α1/β mRNA expression. → L pro inhibits MDA5-mediated activation of the IFN-α1/β promoter. → L pro significantly reduced the transcription of multiple IRF-responsive genes. → L pro inhibits IFN-α1/β promoter activation by decreasing IRF-3/7 in protein levels. → The ability to process eIF-4G of L pro is not necessary to inhibit IFN-α1/β activation. -- Abstract: The leader proteinase (L pro ) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-β (IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor κB (NF-κB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-α1/β expression caused by L pro was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-α/β. Furthermore, overexpression of L pro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L pro mutants indicated that the ability to process eIF-4G of L pro is not required for suppressing dsRNA-induced activation of the IFN-α1/β promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-κB, L pro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

Energy Technology Data Exchange (ETDEWEB)

Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

2010-08-13

Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

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.

A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

Energy Technology Data Exchange (ETDEWEB)

Hickey, E.W.

1988-01-01

Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH{sub 0}) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH{sub 0} induced behavior in E. coli and S. typhimurium by quantitation of H{sub 2} {sup 35}SO{sub 4} labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH{sub 0} 5.0 or shifted from growth at pH{sub 0} 7.0 to 5.0. Internal pH (pH{sub i}) changes occurring during steady state growth at low pH{sub 0}, and on shifting from pH{sub 0} 7.0 to 5.0, were measured using {sup 14}C-benzoic acid uptake.

Globalization and sustainable development an E7 point of view

Energy Technology Data Exchange (ETDEWEB)

Strassburg, W.

1998-07-01

The energy sector is a crucial feedstock to economic development. One example of this industry sector's contribution towards sustainable development is the so-called E7 initiative on sustainable energy development of some of the biggest electric utilities. E7 members from Canada, France, Germany, Italy, Japan and the US are demonstrating the functioning of the requested globally synchronized approach between developed and developing countries. E7 members focus their activities on (1) the efficient use of primary energy resources, (2) maximizing the use of renewable energy resources, (3) maximizing efficiency in the generation, delivery, and use of electricity, (4) minimizing environmental impacts of energy production and use and, (5) implementing innovative options to promote win-win benefits for the environment and the economy through their network of expertise on a pro bono basis. A main emphasis lies in the consistent Joint Implementation given the fact that innovative and efficient Green House Gas reduction measures will be the core of future E7 activities. Especially commercial projects will provide valuable contributions to Green House Gas reductions as well as to economic development of the recipient country. Other instruments, such Emission Trading and Clean Development Mechanism will have to be investigated appropriately in terms of their practicability for Green House Gas reductions so that their will be a notification by the international climate protection regime. Therefore Emission Trading and Clean Development Mechanism are of importance for E7 members' future operations, too. With respect to Sustainable Development industry, energy and environmental policy in leading industry countries must be more closely aligned to a global approach than has so far been the case. The electricity sector is called on to make a major contribution.

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

Energy Technology Data Exchange (ETDEWEB)

Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T. (Biosciences Division); ( XSD); ( PSC-USR); (Univ. of Illinois at Chicago); (Univ. of Minnesota)

2010-09-09

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

Directory of Open Access Journals (Sweden)

Inge Van der Linden

2016-01-01

Full Text Available Numerous outbreaks of Escherichia coli O157:H7 have been linked to the consumption of leafy vegetables. However, up to the present, little has been known about E. coli O157:H7’s adaptive responses to survival on actively growing (and thus responsive plants. In this study, whole genome transcriptional profiles were generated from E. coli O157:H7 cells (isolate Sakai, stx- one hour and two days after inoculation on the leaves of growing butterhead lettuce, and compared with an inoculum control. A total of 273 genes of E. coli O157:H7 Sakai (5.04% of the whole genome were significantly induced or repressed by at least two-fold (p < 0.01 in at least one of the analyzed time points in comparison with the control. Several E. coli O157:H7 genes associated with oxidative stress and antimicrobial resistance were upregulated, including the iron-sulfur cluster and the multiple antibiotic resistance (mar operon, whereas the Shiga toxin virulence genes were downregulated. Nearly 40% of the genes with significantly different expression were poorly characterized genes or genes with unknown functions. These genes are of special interest for future research as they may play an important role in the pathogens’ adaptation to a lifestyle on plants. In conclusion, these findings suggest that the pathogen actively interacts with the plant environment by adapting its metabolism and responding to oxidative stress.

Novel functions for atypical E2Fs, E2F7 and E2F8, in polyploidization and liver cancer

NARCIS (Netherlands)

Pandit, Shusil Kumar

2014-01-01

Atypical E2F transcription factors, E2F7 and E2F8, function as transcriptional repressors of E2F target genes and are crucial for controlling the cell proliferation. In this thesis, we reveal that these two factors are crucial for liver cell polyploidization, embryonic development and prevention of

A novel cis-acting element required for DNA damage-inducible expression of yeast DIN7

International Nuclear Information System (INIS)

Yoshitani, Ayako; Yoshida, Minoru; Ling Feng

2008-01-01

Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified trans-acting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage

NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

Energy Technology Data Exchange (ETDEWEB)

Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: shakim@lgls.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)

2012-08-15

Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

Lactobacillus acidophilus ameliorates H. pylori-induced gastric inflammation by inactivating the Smad7 and NFκB pathways

Directory of Open Access Journals (Sweden)

Yang Yao-Jong

2012-03-01

Full Text Available Abstract Background H. pylori infection may trigger Smad7 and NFκB expression in the stomach, whereas probiotics promote gastrointestinal health and improve intestinal inflammation caused by pathogens. This study examines if probiotics can improve H. pylori-induced gastric inflammation by inactivating the Smad7 and NFκB pathways. Results Challenge with H. pylori increased IL-8 and TNF-α expressions but not TGF-β1 in MKN45 cells. The RNA levels of Smad7 in AGS cells increased after H. pylori infection in a dose-dependent manner. A higher dose (MOI 100 of L. acidophilus pre-treatment attenuated the H. pylori-induced IL-8 expressions, but not TGF-β1. Such anti-inflammatory effect was mediated via increased cytoplasmic IκBα and depletion of nuclear NFκB. L. acidophilus also inhibited H. pylori-induced Smad7 transcription by inactivating the Jak1 and Stat1 pathways, which might activate the TGF-β1/Smad pathway. L. acidophilus pre-treatment ameliorated IFN-γ-induced Smad7 translation level and subsequently reduced nuclear NF-κB production, as detected by western blotting. Conclusions H. pylori infection induces Smad7, NFκB, IL-8, and TNF-α production in vitro. Higher doses of L. acidophilus pre-treatment reduce H. pylori-induced inflammation through the inactivation of the Smad7 and NFκB pathways.

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.

Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

Science.gov (United States)