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Sample records for cell proviral dna

  1. Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders

    International Nuclear Information System (INIS)

    Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, the authors have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. They have clones the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome

  2. The proviral genome of radiation leukemia virus (RadLV): molecular cloning, restriction analysis and integration sites in tumor cell DNA

    International Nuclear Information System (INIS)

    An infectious clone of the linear, unintegrated RadLV provirus was obtained by insertion in the plasmid pBR322. Its restriction map was indistinguishable from that of the majority of the multiple proviral copies, which are found apparently at random sites in the DNA of RadLV-induced rat thymic lymphomas

  3. Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA

    DEFF Research Database (Denmark)

    Jakobsen, Martin Roelsgaard; Tolstrup, M; Bertelsen, L;

    2007-01-01

    BACKGROUND: Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO(+)T cell compartment. OBJECTIVE: To develop an ARMS assay for plasma virions and to...... investigate the expression of resistance mutations (103N and 184V) and dynamic interactions between proviral DNA and plasma virions. STUDY DESIGN: A clinical cross-sectional study, including 11 patients on lamivudine efavirenz and/or nevirapine therapy. The viral populations were determined by an assay based...

  4. Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

    International Nuclear Information System (INIS)

    Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-γ enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus

  5. Coamplification of HIV-1 Proviral DNA and Viral RNA in Assays Used for Quantification of HIV-1 RNA▿

    OpenAIRE

    Wan, H; Seth, A; Rainen, L; Fernandes, H.

    2010-01-01

    Elevated HIV-1 viral load (VL) observed in specimens frozen in situ in plasma preparation tubes (PPTs) compared to EDTA plasma specimens may affect therapeutic monitoring of HIV-infected patients. The increase in viral load is cell associated and minimized when plasma from the PPT is aspirated or recentrifuged prior to freezing. This study investigates the contribution of integrated HIV-1 proviral DNA to elevated VL in the quantification of HIV-1 RNA in plasma. Fifty paired specimens collecte...

  6. Investigation of the bovine leukemia virus proviral DNA in human leukemias and lung cancers in Korea.

    Science.gov (United States)

    Lee, Jehoon; Kim, Yonggoo; Kang, Chang Suk; Cho, Dae Hyun; Shin, Dong Hwan; Yum, Young Na; Oh, Jae Ho; Kim, Sheen Hee; Hwang, Myung Sil; Lim, Chul Joo; Yang, Ki Hwa; Han, Kyungja

    2005-08-01

    The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans. PMID:16100451

  7. The proviral genome of radiation leukemia virus: Molecular cloning, nucleotide sequence of its long terminal repeat and integration in lymphoma cell DNA

    International Nuclear Information System (INIS)

    The proviral genome of a thymotropic and leukemogenic C57BL/Ka mouse retrovirus, RadLV/VL/sub 3/(T+L+), was cloned as a biologically active PstI insert in the bacterial plasmid pBR322. Its restriction map was compared to those, already known, of two nonthymotropic and nonleukemogenic viruses of the same mouse strain, the ecotropic BL/Ka(B) and the xenotropic constituent of the radiation leukemia virus complex (RadLV). Differences were observed in the pol gene and in the env gene. Moreover, the nucleotide sequence of the RadLV/VL/sub 3/(T+L+) long terminal repeat revealed the existence of two copies of a 42 bp long sequence, separated by 11 nucleotides and of which BL/Ka(B) possesses only one copy

  8. Comparative genotypic study of viral RNA and proviral DNA in HIV-1 patients

    OpenAIRE

    Chan, Wing-tsin, Alison; 陳穎芊

    2015-01-01

    Highly Active Antiretroviral Therapy (HAART) has been used for HIV-1 treatment for more than a decade. Prior identification of drug resistance associated mutations and viral tropism provides valuable information for physicians in optimizing the best HAART regimens for each patient. Plasma RNA has long been used as the major laboratory marker as it reflects the status of circulating virus. However, it might not be as informative in patients with low viral loads. Proviral DNA was therefore sugg...

  9. Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

    Institute of Scientific and Technical Information of China (English)

    YIN Qian Qian; SHAO Yi Ming; MA Li Ying; LI Zhen Peng; ZHAO Hai; PAN Dong; WANG Yan; XU Wei Si; XING Hui; FENGYi; JIANG Shi Bo

    2016-01-01

    ObjectiveTo investigate distinctive features in drug-resistant mutations(DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients. MethodsForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. ResultsCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher’s exact test,P ConclusionCompared with viral RNA, the distinctive information of DRMsand drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

  10. Investigating signs of recent evolution in the pool of proviral HIV type 1 DNA during years of successful HAART

    DEFF Research Database (Denmark)

    Mens, Helene; Pedersen, Anders G; Jørgensen, Louise B;

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene, was...... determine whether there were temporal trends indicating ongoing replication and evolution. In summary, it was not possible to detect definitive signs of ongoing evolution in either the bulk-sequenced or the clonal data with the methods employed here, but our results could be consistent with localized...... expression of archival HIV genomes in some patients. Interestingly, stop-codons were present at the same two positions in several independent clones and across patients. Simulation studies indicated that this phenomenon could be explained as the result of parallel evolution and that some sites were...

  11. Investigating Signs of Recent Evolution in the Pool of Pro-viral DNA during Years of Successful HAART

    DEFF Research Database (Denmark)

    Mens, H.; Pedersen, Anders Gorm; Jørgensen, L. B.;

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene, was...... determine whether there were temporal trends indicating ongoing replication and evolution. In summary, it was not possible to detect definitive signs of ongoing evolution in either the bulk-sequenced or the clonal data with the methods employed here, but our results could be consistent with localized...... expression of archival HIV genomes in some patients. Interestingly, stop-codons were present at the same two positions in several independent clones and across patients. Simulation studies indicated that this phenomenon could be explained as the result of parallel evolution and that some sites were...

  12. Investigating signs of recent evolution in the pool of proviral HIV type 1 DNA during years of successful HAART

    DEFF Research Database (Denmark)

    Mens, Helene; Pedersen, Anders G; Jørgensen, Louise B;

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene, was...... collected from PBMCs isolated from 57 patients. Both "consensus" (57 patients) and clonal (7 patients) sequences were obtained from five time points spanning a 24-month period. The main computational strategy was to use maximum likelihood to fit a set of alternative phylogenetic models to the clonal data......, and then determine the support for models that imply evolution between time points. Model fit and model-selection uncertainty was assessed using the Akaike information criterion (AIC) and Akaike weights. The consensus sequence data was also analyzed using a range of phylogenetic techniques to...

  13. Results of External Quality Assessment for Proviral DNA Testing of HIV Tropism in the Maraviroc Switch Collaborative Study

    OpenAIRE

    Tu, Elise; Swenson, Luke C.; Land, Sally; Pett, Sarah; Emery, Sean; Marks, Kat; Kelleher, Anthony D.; Kaye, Steve; Kaiser, Rolf; Schuelter, Eugene; Harrigan, Richard

    2013-01-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination usin...

  14. Genotypic tropism testing in proviral DNA to guide maraviroc initiation in aviremic subjects: 48-week analysis of the PROTEST study

    Directory of Open Access Journals (Sweden)

    Federico Garcia

    2014-11-01

    Full Text Available Introduction: In a previous interim 24-week virological safety analysis of the PROTEST study (1, initiation of Maraviroc (MVC plus 2 nucleoside reverse-transcriptase inhibitors (NRTIs in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA was associated with low rates of virological failure. Here we present the final 48-week analysis of the study. Methods: PROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910 carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL 10% in a singleton, initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week*100. Results: Tropism results were available from 141/175 (80.6% subjects screened: 87/141 (60% were R5 and 74/87 (85% were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62% subjects, NNRTIs in 27 (36% and integrase inhibitors (INIs in 1 (2%. The main reasons for treatment change were dyslipidemia (42%, gastrointestinal symptoms (22%, and liver toxicity (15%. MVC was given alongside TDF/FTC in 40 (54% subjects, ABC/3TC in 30 (40%, AZT/3TC in 2 (3% and ABC/TDF in 2 (3%. Sixty-two (84% subjects maintained VL<50 c/mL through week 48, whereas 12 (16% discontinued treatment: two (3% withdrew informed consent, one (1% had a R5→X4 shift in HIV tropism between the screening and baseline visits, one (1% was lost to follow-up, one (1% developed an ART-related adverse event (rash, two (3% died due to non-study-related causes (1 myocardial infarction at week 0 and 1 lung cancer at week 36, and five (7% developed protocol

  15. Complete Sequence of Proviral DNA of Equine Infectious Anemia Virus Strain L

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-quan; WANG Liu; YANG Zhi-biao; KONG Xian-gang; TONG Guang-Zhi

    2002-01-01

    Equine infectious anemia virus strain L (EIAV-L) is the parental virulent virus of equine infectious anemia donkey leukocyte attenuated vaccine (DLA EIAV ). In this study, peripheral blood leukocytes(PBL) were collected from a horse infected with EIAV-L. The PBL DNAs were extracted. The EIAV-L proviral DNA was amplified in four parts covering the entire proviral genomic sequence by polymerase chain reaction (PCR). Each of the four parts was cloned into the plasmid pBluescript SK, and the recombinant plasmids were designated as p2.8, p2.4, p3.1, and p1.2 respectively. After identification with restriction digestion, the inserts within the four plasmids were sequenced. The complete nucleotide sequence of EIAV-L provirus was determined by analyzing each of the four parts and connecting them as a whole. The genome of EIAV-L is 8235 bp in length, and G + C content is 38%. The comparison analysis by the computer software DNASIS showed that the sequence of EIAV-L shares 98.4% and 96.9% identities with that of D-A EIAV and DLA EIAV respectively. The high homology between these strains showed that they were genetically related.The homology between EIAV-L and D-A EIAV is higher than that between EIAV-L and DLA EIAV, and this is consistent with the derivation progress of DLA EIAV. At both ends of EIAV-L provirus, there is an identical long terminal repeat (LTR) sequence of 316bp in length. The LTR consists of U3, R, and U5 regions. The genome of EIAV-L provirus has three long open reading frames(ORF) corresponding to gag, pol and env genes respectively. The gag gene is 1200bp and located at position 613-1912nt. The pol gene is 3402bp and located at position 1708-5109nt. There is a termination codon within the env dividing it into two parts,env1 of 699bp (position 5305-6003nt)and env2 of 1827bp (position 6073-7899nt). The provirus has three additional small ORFs: S1, S2 and S3 with sizes of 153bp (position 5113-5265nt), 204bp (position 5279-5482nt) and 402bp ( position 7245

  16. Human T-lymphotropic virus type 1 (HTLV-1 prevalence and quantitative detection of DNA proviral load in individuals with indeterminate/positive serological results

    Directory of Open Access Journals (Sweden)

    Bon Isabella

    2006-03-01

    Full Text Available Abstract Background HTLV-1 infection is currently restricted to endemic areas. To define the prevalence of HTLV-1 infection in patients living in Italy, we first carried out a retrospective serological analysis in a group of people originating from African countries referred to our hospital from January 2003 to February 2005. We subsequently applied a real time PCR on peripheral blood mononuclear cells from subjects with positive or indeterminate serological results. Methods All the sera were first analysed by serological methods (ELISA and/or Western Blotting and then the peripheral blood mononuclear cells from subjects with positive or inconclusive serological results were analyzed for the presence of proviral DNA by a sensitive SYBR Green real time PCR. In addition, twenty HTLV-I ELISA negative samples were assayed by real time PCR approach as negative controls. Results Serological results disclosed serum reactivity by ELISA (absorbance values equal or greater than the cut-off value in 9 out of 3408 individuals attending the Sexually Transmitted Diseases Clinic and/or Oncology Department, and 2 out 534 blood donors enrolled as a control population. Irrespective of positive or inconclusive serological results, all these subjects were analyzed for the presence of proviral DNA in peripheral blood mononuclear cells by SYBR real time PCR. A clear-cut positive result for the presence of HTLV-1 DNA was obtained in two subjects from endemic areas. Conclusion SYBR real time PCR cut short inconclusive serological results. This rapid and inexpensive assay showed an excellent linear dynamic range, specificity and reproducibility readily revealing and quantifying the presence of virus in PBMCs. Our results highlight the need to monitor the presence of HTLV-1 in countries which have seen a large influx of immigrants in recent years. Epidemiological surveillance and correct diagnosis are recommended to verify the prevalence and incidence of a new

  17. Proviral genome of radiation leukemia virus: molecular cloning of biologically active proviral DNA and nucleotide sequence of its long terminal repeat.

    OpenAIRE

    Janowski, M; Merregaert, J; Boniver, J; Maisin, J R

    1985-01-01

    The proviral genome of a leukemogenic and thymotropic C57BL/Ka mouse retrovirus, RadLV/VL3(T+L+), was cloned as a biologically active PstI insert in the bacterial plasmid pBR322. Its restriction map was compared with those, already known, of two nonthymotropic and nonleukemogenic viruses of the same mouse strain: the ecotropic BL/Ka(B) virus and the xenotropic constituent of the radiation leukemia virus complex. Differences were observed around the gag-pol gene junction, in the pol gene, and ...

  18. The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA

    OpenAIRE

    Zhu, Weijun; Lei, Rongyue; Le Duff, Yann; Li, Jian; Guo, Fei; Wainberg, Mark A.; Liang, Chen

    2015-01-01

    Background Highly active antiretroviral therapy (HAART) has transformed HIV-1 infection from a deadly disease to a manageable chronic illness, albeit does not provide a cure. The recently developed genome editing system called CRISPR/Cas9 offers a new tool to inactivate the integrated latent HIV-1 DNA and may serve as a new avenue toward cure. Findings We tested 10 sites in HIV-1 DNA that can be targeted by CRISPR/Cas9. The engineered CRISPR/Cas9 system was introduced into the JLat10.6 cells ...

  19. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    Science.gov (United States)

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  20. Toll-Interacting Protein Suppresses HIV-1 Long-Terminal-Repeat-Driven Gene Expression and Silences the Post-Integrational Transcription of Viral Proviral DNA.

    Directory of Open Access Journals (Sweden)

    Fu-Chun Yang

    Full Text Available Toll-interacting protein (Tollip is a host adaptor protein for negatively regulating Toll-like receptor 2-, 4-, and IL-1R (interleukin-1 receptor-mediated signaling. We found that Tollip expression could be induced in MDDCs (monocyte-derived dendritic cells by HIV-1 particles and recombinant gp120 glycoprotein. Hence, we investigated the role of Tollip in modulating HIV-1 infection. We found that Tollip expression suppressed NF-κB-dependent HIV-1 long terminal repeat (LTR-driven transcription and thus inhibited HIV-1 infection. Our protein truncation experiments proved that the intact C-terminus of Tollip was required for inhibition of both NF-κB activity and HIV-1 LTR-driven gene expression. Intriguingly, Tollip silenced the post-integrational transcription of HIV-1 proviral DNA, indicating the potential role of Tollip in maintaining viral persistence. Our results reveal the novel role of host factor Tollip in modulating HIV-1 infection, and may suggest the hijacking of Tollip as the negative regulator of the TLR pathway and even the downstream signaling, by HIV-1 for maintaining persistent infection. Further elucidation of the mechanisms by which HIV-1 induces Tollip expression and identification of the role of Tollip in modulating HIV-1 latency will facilitate the understanding of host regulation in viral replication and benefit the exploration of novel strategies for combating HIV-1 infection.

  1. Quantification of HTLV-I proviral load in experimentally infected rabbits

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    Kindt Thomas J

    2005-05-01

    Full Text Available Abstract Background Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection. Results A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1. Conclusion Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals.

  2. Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells.

    OpenAIRE

    Hynes, N E; Kennedy, N; Rahmsdorf, U.; Groner, B.

    1981-01-01

    A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferas...

  3. Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea

    OpenAIRE

    Lee, JeHoon; Kim, Yonggoo; Kang, Chang Suk; Cho, Dae Hyun; Shin, Dong Hwan; Yum, Young Na; Oh, Jae Ho; Kim, Sheen Hee; Hwang, Myung Sil; Lim, Chul Joo; Yang, Ki Hwa; Han, Kyungja

    2005-01-01

    The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combinati...

  4. Transcriptional suppression of in vitro-integrated human immunodeficiency virus type 1 does not correlate with proviral DNA methylation

    Czech Academy of Sciences Publication Activity Database

    Pion, M.; Jordan, A.; Biancotto, A.; Dequiedt, F.; Gondois-Rey, F.; Rondeau, S.; Vigne, R.; Hejnar, Jiří; Verdin, E.; Hirsch, I.

    2003-01-01

    Roč. 77, č. 7 (2003), s. 4025-4032. ISSN 0022-538X R&D Projects: GA ČR GA204/01/0632 Institutional research plan: CEZ:AV0Z5052915 Keywords : HIV-1 latency * transcriptional suppression * proviral CpG methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.225, year: 2003

  5. Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis

    OpenAIRE

    MIURA, Saori; HORIUCHI, Noriyuki; MATSUMOTO, Kotaro; Kobayashi, Yoshiyasu; Kawazu, Shin-ichiro; INOKUMA, Hisashi

    2015-01-01

    Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with ...

  6. Administration of a Toll-like receptor 9 agonist decreases the proviral reservoir in virologically suppressed HIV-infected patients.

    Directory of Open Access Journals (Sweden)

    Anni A Winckelmann

    Full Text Available Toll-like receptor (TLR agonists can reactivate HIV from latently infected cells in vitro. We aimed to investigate the TLR-9 agonist, CPG 7909's in vivo effect on the proviral HIV reservoir and HIV-specific immunity. This was a post-hoc analysis of a double-blind randomized controlled vaccine trial. HIV-infected adults were randomized 1:1 to receive pneumococcal vaccines with or without 1 mg CPG 7909 as adjuvant at 0, 3 and 9 months. In patients on suppressive antiretroviral therapy we quantified proviral DNA at 0, 3, 4, 9, and 10 months (31 subjects in the CPG group and 37 in the placebo-adjuvant group. Furthermore, we measured HIV-specific antibodies, characterized T cell phenotypes and HIV-specific T cell immunity. We observed a mean reduction in proviral DNA in the CPG group of 12.6% (95% CI: -23.6-0.0 following each immunization whereas proviral DNA in the placebo-adjuvant group remained largely unchanged (6.7% increase; 95% CI: -4.2-19.0 after each immunization, p = 0.02. Among participants with additional cryo-preserved PBMCs, HIV-specific CD8+ T cell immunity as indicated by increased expression of degranulation marker CD107a and macrophage inflammatory protein 1β (MIP1β tended to be up-regulated following immunization with CPG 7909 compared with placebo as adjuvant. Further, increasing proportion of HIV-specific CD107a and MIP1β-expressing CD8+ T cells were strongly correlated with decreasing proviral load. No changes were observed in T cell phenotype distribution, HIV-specific CD4+ T cell immunity, or HIV-specific antibodies. TLR9-adjuvanted pneumococcal vaccination decreased proviral load. Reductions in proviral load correlated with increasing levels of HIV specific CD8+ T cells. Further investigation into the potential effect of TLR9 agonists on HIV latency is warranted.

  7. Administration of a Toll-like receptor 9 agonist decreases the proviral reservoir in virologically suppressed HIV-infected patients.

    Science.gov (United States)

    Winckelmann, Anni A; Munk-Petersen, Lærke V; Rasmussen, Thomas A; Melchjorsen, Jesper; Hjelholt, Thomas J; Montefiori, David; Østergaard, Lars; Søgaard, Ole S; Tolstrup, Martin

    2013-01-01

    Toll-like receptor (TLR) agonists can reactivate HIV from latently infected cells in vitro. We aimed to investigate the TLR-9 agonist, CPG 7909's in vivo effect on the proviral HIV reservoir and HIV-specific immunity. This was a post-hoc analysis of a double-blind randomized controlled vaccine trial. HIV-infected adults were randomized 1:1 to receive pneumococcal vaccines with or without 1 mg CPG 7909 as adjuvant at 0, 3 and 9 months. In patients on suppressive antiretroviral therapy we quantified proviral DNA at 0, 3, 4, 9, and 10 months (31 subjects in the CPG group and 37 in the placebo-adjuvant group). Furthermore, we measured HIV-specific antibodies, characterized T cell phenotypes and HIV-specific T cell immunity. We observed a mean reduction in proviral DNA in the CPG group of 12.6% (95% CI: -23.6-0.0) following each immunization whereas proviral DNA in the placebo-adjuvant group remained largely unchanged (6.7% increase; 95% CI: -4.2-19.0 after each immunization, p = 0.02). Among participants with additional cryo-preserved PBMCs, HIV-specific CD8+ T cell immunity as indicated by increased expression of degranulation marker CD107a and macrophage inflammatory protein 1β (MIP1β) tended to be up-regulated following immunization with CPG 7909 compared with placebo as adjuvant. Further, increasing proportion of HIV-specific CD107a and MIP1β-expressing CD8+ T cells were strongly correlated with decreasing proviral load. No changes were observed in T cell phenotype distribution, HIV-specific CD4+ T cell immunity, or HIV-specific antibodies. TLR9-adjuvanted pneumococcal vaccination decreased proviral load. Reductions in proviral load correlated with increasing levels of HIV specific CD8+ T cells. Further investigation into the potential effect of TLR9 agonists on HIV latency is warranted. PMID:23637967

  8. Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis.

    Science.gov (United States)

    Miura, Saori; Horiuchi, Noriyuki; Matsumoto, Kotaro; Kobayashi, Yoshiyasu; Kawazu, Shin-Ichiro; Inokuma, Hisashi

    2015-07-01

    Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle. PMID:25766769

  9. The Icsbp locus is a common proviral insertion site in mature B-cell lymphomas/plasmacytomas induced by exogenous murine leukemia virus

    International Nuclear Information System (INIS)

    ICSBP (interferon consensus sequence binding protein)/IRF8 (interferon regulatory factor 8) is an interferon gamma-inducible transcription factor expressed predominantly in hematopoietic cells, and down-regulation of this factor has been observed in chronic myelogenous leukemia and acute myeloid leukemia in man. By screening about 1200 murine leukemia virus (MLV)-induced lymphomas, we found proviral insertions at the Icsbp locus in 14 tumors, 13 of which were mature B-cell lymphomas or plasmacytomas. Only one was a T-cell lymphoma, although such tumors constituted about half of the samples screened. This indicates that the Icsbp locus can play a specific role in the development of mature B-lineage malignancies. Two proviral insertions in the last Icsbp exon were found to act by a poly(A)-insertion mechanism. The remaining insertions were found within or outside Icsbp. Since our results showed expression of Icsbp RNA and protein in all end-stage tumor samples, a simple tumor suppressor function of ICSBP is not likely. Interestingly, proviral insertions at Icsbp have not been reported from previous extensive screenings of mature B-cell lymphomas induced by endogenous MLVs. We propose that ICSBP might be involved in an early modulation of an immune response to exogenous MLVs that might also play a role in proliferation of the mature B-cell lymphomas

  10. Risk factors associated with increased bovine leukemia virus proviral load in infected cattle in Japan from 2012 to 2014.

    Science.gov (United States)

    Ohno, Ayumu; Takeshima, Shin-nosuke; Matsumoto, Yuki; Aida, Yoko

    2015-12-01

    Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, a malignant B cell lymphoma. BLV has spread worldwide and causes serious problems. After infection, the BLV genome is integrated into the host DNA and can be amplified during periods of latency. We previously designed degenerate primers using the Coordination of Common Motifs (CoCoMo) algorithm to establish a new quantitative real-time PCR method (BLV-CoCoMo-qPCR-2) of measuring the proviral load of both known and novel BLV variants. Here, we aimed to examine the correlation between proviral load and risk factors for BLV infection, such as breeding systems, parousity, and colostrum feeding. Blood and serum samples were collected from 83 BLV-positive farms in 22 prefectures of Japan, and the BLV proviral load and anti-BLV antibody levels were measured. BLV was detected in 73.3% (1039/1,417) of cattle by BLV-CoCoMo-qPCR-2 and the provirus was detected in 93 of 1039 antibody-negative samples. The results showed that the proviral load increased with progression of lymphocytosis. Next, the risk factors associated with increasing BLV infection rate were examined along with any association with proviral load. The proviral load was higher in cattle with lymphocytosis than in healthy cattle, and higher in multiparous cows than in nulliparous cows. Finally, proviral loads were higher in contact breeding systems than in non-contact breeding systems. Taken together, these findings may help to formulate a plan for eliminating BLV from contaminated farms. This is the first nationwide study to estimate BLV proviral load in Japanese cattle. PMID:26321160

  11. Dynamic interaction between STLV-1 proviral load and T-cell response during chronic infection and after immunosuppression in non-human primates.

    Directory of Open Access Journals (Sweden)

    Sandrine Souquière

    Full Text Available We used mandrills (Mandrillus sphinx naturally infected with simian T-cell leukemia virus type 1 (STLV-1 as a model for evaluating the influence of natural STLV-1 infection on the dynamics and evolution of the immune system during chronic infection. Furthermore, in order to evaluate the role of the immune system in controlling the infection during latency, we induced immunosuppression in the infected monkeys. We first showed that the STLV-1 proviral load was higher in males than in females and increased significantly with the duration of infection: mandrills infected for 10-6 years had a significantly higher proviral load than those infected for 2-4 years. Curiously, this observation was associated with a clear reduction in CD4+ T-cell number with age. We also found that the percentage of CD4(+ T cells co-expressing the activation marker HLA-DR and the mean percentage of CD25(+ in CD4(+ and CD8(+ T cells were significantly higher in infected than in uninfected animals. Furthermore, the STLV-1 proviral load correlated positively with T-cell activation but not with the frequency of T cells secreting interferon gamma in response to Tax peptides. Lastly, we showed that, during immunosuppression in infected monkeys, the percentages of CD8(+ T cells expressing HLA-DR(+ and of CD4(+ T cells expressing the proliferation marker Ki67 decreased significantly, although the percentage of CD8(+ T cells expressing HLA-DR(+ and Ki67 increased significantly by the end of treatment. Interestingly, the proviral load increased significantly after immunosuppression in the monkey with the highest load. Our study demonstrates that mandrills naturally infected with STLV-1 could be a suitable model for studying the relations between host and virus. Further studies are needed to determine whether the different compartments of the immune response during infection induce the long latency by controlling viral replication over time. Such studies would provide important

  12. Multiple proviral integration events after virological synapse-mediated HIV-1 spread

    Energy Technology Data Exchange (ETDEWEB)

    Russell, Rebecca A., E-mail: rebecca.moore@path.ox.ac.uk [The Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX13RE (United Kingdom); Martin, Nicola; Mitar, Ivonne [The Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX13RE (United Kingdom); Jones, Emma [The Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, Wales (United Kingdom); Sattentau, Quentin J. [The Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX13RE (United Kingdom)

    2013-08-15

    HIV-1 can move directly between T cells via virological synapses (VS). Although aspects of the molecular and cellular mechanisms underlying this mode of spread have been elucidated, the outcomes for infection of the target cell remain incompletely understood. We set out to determine whether HIV-1 transfer via VS results in productive, high-multiplicity HIV-1 infection. We found that HIV-1 cell-to-cell spread resulted in nuclear import of multiple proviruses into target cells as seen by fluorescence in-situ hybridization. Proviral integration into the target cell genome was significantly higher than that seen in a cell-free infection system, and consequent de novo viral DNA and RNA production in the target cell detected by quantitative PCR increased over time. Our data show efficient proviral integration across VS, implying the probability of multiple integration events in target cells that drive productive T cell infection. - Highlights: • Cell-to-cell HIV-1 infection delivers multiple vRNA copies to the target cell. • Cell-to-cell infection results in productive infection of the target cell. • Cell-to-cell transmission is more efficient than cell-free HIV-1 infection. • Suggests a mechanism for recombination in cells infected with multiple viral genomes.

  13. Multiple proviral integration events after virological synapse-mediated HIV-1 spread

    International Nuclear Information System (INIS)

    HIV-1 can move directly between T cells via virological synapses (VS). Although aspects of the molecular and cellular mechanisms underlying this mode of spread have been elucidated, the outcomes for infection of the target cell remain incompletely understood. We set out to determine whether HIV-1 transfer via VS results in productive, high-multiplicity HIV-1 infection. We found that HIV-1 cell-to-cell spread resulted in nuclear import of multiple proviruses into target cells as seen by fluorescence in-situ hybridization. Proviral integration into the target cell genome was significantly higher than that seen in a cell-free infection system, and consequent de novo viral DNA and RNA production in the target cell detected by quantitative PCR increased over time. Our data show efficient proviral integration across VS, implying the probability of multiple integration events in target cells that drive productive T cell infection. - Highlights: • Cell-to-cell HIV-1 infection delivers multiple vRNA copies to the target cell. • Cell-to-cell infection results in productive infection of the target cell. • Cell-to-cell transmission is more efficient than cell-free HIV-1 infection. • Suggests a mechanism for recombination in cells infected with multiple viral genomes

  14. Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing

    OpenAIRE

    Leung, Danny C.; Dong, Kevin B; Maksakova, Irina A.; Goyal, Preeti; Appanah, Ruth; Lee, Sandra; Tachibana, Makoto; Shinkai, Yoichi; Lehnertz, Bernhard; Mager, Dixie L; Rossi, Fabio; Lorincz, Matthew C.

    2011-01-01

    Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, des...

  15. Detection and quantification of proviral HIV-1 184 M/V in circulating CD4(+) T cells of patients on HAART with a viremia less than 1000 copies/ml

    DEFF Research Database (Denmark)

    Mohey, Rajesh; Jørgensen, Anne Louise; Møller, Bjarne K;

    2005-01-01

    Background Highly active anti-retroviral therapy (HAART) effectively reduces HIV replication but does not completely hinder it. Sub-optimal therapy leads to HIV resistance to the drugs administered. However, the role of low-level viremia (viral-load less than 1000 copies/ml) on mutation genesis and...... proviral HIV-1 was detected and quantified by a specific and sensitive assay combining a TaqMan real-time PCR analysis with the amplification-refractory mutation system (ARMS) principle. Results Fifty-six percent of patients with low-level viremia had 184V in the CD4+ T cellular DNA compartment as compared...... had a median viral load of 498 copies/ml (P = 0.006). No significant differences between the groups were observed in proviral HIV-1 DNA load. Conclusions The frequency of the 184V mutation was significantly lower, in the CD4+ T cellular compartment of patients with a viral load of less than 20 copies...

  16. DNA-cell conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  17. E box motifs as mediators of proviral latency of human retroviruses

    Directory of Open Access Journals (Sweden)

    Gazzolo Louis

    2009-09-01

    Full Text Available Abstract The palindromic sequence motifs (CANNTG known as E boxes are considered as binding sites for the basic helix-loop-helix (bHLH class of DNA-binding proteins. Their presence has been reported in the long terminal repeats (LTR of the HIV-1 and HTLV-1 proviruses. Their close proximity with the TATA region of both LTRs indicates that the bHLH proteins may act as important regulators of the function of proviral transcription. Indeed, observations on HIV-1 and recent results on HTLV-1 underline that these E boxes may be critically involved in the regulation of the proviral transcription of these human retroviruses. Indeed, of the two E boxes flanking the TATA sequences of the HIV-1 provirus, the 3' E box has been implicated in the transcriptional inhibition of viral gene expression. Such a role might also be played by the unique 5' E box present in the HTLV-1 LTR. In both cases, the expression of tissue-specfic bHLH proteins, like TAL1 might counteract the inhibitory effect exerted by E box proteins, thereby increasing proviral transcription. Finally, a phylogenetic study encompassing several subtypes of these two human retroviruses underlines that these E box motifs have recently appeared in the proviral LTRs and may be considered as potential mediators in the establishment of proviral latency.

  18. Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

    the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells and latent HIV-infected cell lines. Conclusion LV-2xZifHIV-polFN and LV- 2xZifHIV-1FN may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action or disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.

  19. Low proviral small ruminant lentivirus load as biomarker of natural restriction in goats.

    Science.gov (United States)

    Crespo, Helena; Bertolotti, Luigi; Proffiti, Margherita; Cascio, Paolo; Cerruti, Fulvia; Acutis, Pier Luigi; de Andrés, Damián; Reina, Ramsés; Rosati, Sergio

    2016-08-30

    Small ruminant lentiviruses (SRLV) globally affect welfare and production of sheep and goats and are mainly controlled through elimination of infected animals, independently of the viral kinetics within the single animal. Control programs are based on highly sensitive serological tests, however the existence of low antibody responders leads to the permanent presence of seronegative infected animals in the flock, thus perpetuating the infection. On the other hand, long-term non-progressors show a detectable antibody response not indicative of a shedding animal, suggesting immune contention of infection. In this study, we analyse two goat populations within the same herd, harbouring low or high proviral SRLV loads respectively, both showing a robust antibody response. In vivo findings were confirmed in vitro since fibroblastic cell lines obtained from one high and one low proviral load representative goats, showed respectively a high and a faint production of virus upon infection with reference and field circulating SRLV strains. Differences in virus production were relieved when strain CAEV-Co was used for experimental infection. We analysed LTR promoter activity, proviral load, entry step and production of virus and viral proteins. Intriguingly, proteasomal activity was higher in fibroblasts from low proviral load animals and proteasome inhibition increased viral production in both cell lines, suggesting the implication of active proteasome-dependent restriction factors. Among them, we analysed relative expression and sequences of TRIM5α, APOBEC3 (Z1, Z2, Z3 and Z2-Z3) and BST-2 (Tetherin) and found a global antiviral status in low proviral carriers that may confer protection against viral shedding and disease onset. PMID:27527777

  20. HTLV-2 APH-2 Expression Is Correlated With Proviral Load but APH-2 Does Not Promote Lymphocytosis

    OpenAIRE

    Douceron, Estelle; Kaidarova, Zhanna; Miyazato, Paola; Matsuoka, Masao; Murphy, Edward L; Mahieux, Renaud

    2011-01-01

    We recently discovered the antisense protein of human T-cell leukemia virus (HTLV) type 2 (APH-2), whose messenger RNA is encoded by the antisense strand of the HTLV-2 genome. We quantified proviral load, level of tax, and APH-2 in a series of blood samples obtained from a cohort of HTLV-2 carriers. We determined whether APH-2 promotes cell proliferation. APH-2 was detectable in most samples tested and was correlated with proviral load. APH-2 levels were not correlated with lymphocyte count i...

  1. HTLV-2 APH-2 expression is correlated with proviral load but APH-2 does not promote lymphocytosis

    OpenAIRE

    Douceron, E; Kaidarova, Z; Miyazato, P; Matsuoka, M.; Murphy, EL; Mahieux, R.

    2011-01-01

    We recently discovered the antisense protein of human T-cell leukemia virus (HTLV) type 2 (APH-2), whose messenger RNA is encoded by the antisense strand of the HTLV-2 genome. We quantified proviral load, level of tax, and APH-2 in a series of blood samples obtained from a cohort of HTLV-2 carriers. We determined whether APH-2 promotes cell proliferation. APH-2 was detectable in most samples tested and was correlated with proviral load. APH-2 levels were not correlated with lymphocyte count i...

  2. BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

    Directory of Open Access Journals (Sweden)

    Matoba Kazuhiro

    2010-11-01

    Full Text Available Abstract Background Bovine leukemia virus (BLV is closely related to human T-cell leukemia virus (HTLV and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR method using Coordination of Common Motifs (CoCoMo primers to measure the proviral load of known and novel BLV variants in clinical animals. Results Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but

  3. Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901.

    Science.gov (United States)

    Wang, Yu; Cui, Zhizhong; Jiang, Shijin

    2006-04-01

    The genomic DNA extracted from chicken embryo fibroblast (CEF) infected with a Chinese field isolate HA9901 of reticuloendotheliosis virus (REV) was used as the template to amplify the REV proviral genomic cDNA by PCR with 6 pairs of primers according to published sequences. Six overlapping fragments were amplified, cloned into the TA vector and sequenced, including a fragment which was amplified from the circular proviral cDNA and covering both 5'- and 3'-ends. The complete sequence of the whole genome was established and analyzed with a DNAstar software. Comparisons of the sequence with two other strains demonstrated that the genomes of REV were relatively conservative, the homogenecity for all genes or LTR fragments of the 3 strains was over 92%, no matter whether they were isolated from different species and regions in different years. But, the homology of Chinese strain HA9901 to a fowl pox virus-associated strain from Chickens was higher than that to strain SNV isolated from ducks. PMID:16704118

  4. Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

    OpenAIRE

    Giovannangeli, Carine; Diviacco, Silvia; Labrousse, Valérie; Gryaznov, Sergei; Charneau, Pierre; Helene, Claude

    1997-01-01

    The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming olig...

  5. Common proviral integration region on mouse chromosome 7 in lymphomas and myelogenous leukemias induced by Friend murine leukemia virus.

    OpenAIRE

    Silver, J.; Kozak, C

    1986-01-01

    Friend murine leukemia virus (F-MuLV) induces a variety of hematopoietic neoplasms 2 to 12 months after inoculation into newborn mice. These neoplasms are clonal or oligoclonal and contain a small number of F-MuLV insertions in high-molecular-weight DNA. To investigate whether different tumors have proviral insertions in the same region, a provirus-cellular DNA junction fragment from an F-MuLV-induced myelogenous leukemia was cloned in lambda gtWES, and a portion of the flanking cellular DNA ...

  6. Mechanisms of DNA uptake by cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Three categories of cellular uptake of DNA can be distinguished. First, in the highly transformable bacteria, such as Diplococcus pneumoniae, Haemophilus influenzae and Bacillus subtilis, elaborate mechanisms of DNA transport have evolved, presumably for the purpose of genetic exchange. These mechanisms can introduce substantial amounts of DNA into the cell. Second, methods have been devised for the forced introduction of DNA by manipulation of bacterial cells under nonphysiological conditions. By such means small but significant amounts of DNA have been introduced into various bacteria, including Escherichia coli. Third, mammalian cells are able to take up biologically active DNA. This has been most clearly demonstrated with viral DNA, although the mechanism of uptake is not well understood. The intention, here, is to survey current understanding of the various mechanisms of DNA uptake. A review of experience with the bacterial systems may throw some light on the mammalian system and lead to suggestions for enhancing DNA uptake by mammalian cells.

  7. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  8. Biased DNA Segregation during Stem Cell Division

    OpenAIRE

    Anversa, Piero; Leri, Annarosa; Kajstura, Jan

    2012-01-01

    Adult skeletal muscle stem cells are a heterogeneous cell population characterized by a small subset of undifferentiated cells that express at high level the paired/homeodomain gene Pax7. This category of satellite cells divides predominantly by asymmetric chromatid segregation generating a daughter cell that carries the mother DNA and retains stem cell property, and a daughter cell that inherits the newly-synthesized DNA and acquires the myocyte lineage.1

  9. The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1 proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis

    Directory of Open Access Journals (Sweden)

    Chieia Marco

    2008-07-01

    Full Text Available Abstract Background CD4+CD25high regulatory T (TReg cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of TReg cell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of TReg cells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP patients, and to correlate with measures of T cell activation. Results We were able to confirm that HTLV-I drives activation, spontaneous IFNγ production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4+ TReg cells (CD4+CD25high T cells in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4+ TReg cells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127low TReg cells in healthy control subjects. Finally, the proportion of CD127low TReg cells correlated inversely with HTLV-1 proviral load. Conclusion Taken together, the results suggest that TReg cells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4+ T cells, in particular those expressing the CD25highCD127low phenotype. TReg cells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.

  10. DNA charge transport within the cell.

    Science.gov (United States)

    Grodick, Michael A; Muren, Natalie B; Barton, Jacqueline K

    2015-02-01

    The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long-range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long-range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize this chemistry include an interesting and ever-growing class of DNA-processing enzymes involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S clusters. DNA repair enzymes containing 4Fe-4S clusters, that include endonuclease III (EndoIII), MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at long-range via DNA CT. Interactions between DNA and these proteins in solution, in addition to genetics experiments within Escherichia coli, suggest that DNA-mediated CT can be used as a means of cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in finding DNA damage, even within cells. On the basis of these data, we can consider also how DNA-mediated CT may be used as a means of signaling to coordinate DNA processing across the genome. PMID:25606780

  11. Visualization of DNA damage in individual cells

    International Nuclear Information System (INIS)

    A simple technique of micro-agarose gel electrophoresis has been developed to permit an evaluation of DNA damage in individual cells. Cells are embeded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time. In damaged cells, DNA migrated from the nuclei toward the anode, displaying 'comets' visualized by staining with a DNA-specific fluorochrome, acridine orange. The technique was applicable to quantifying DNA damage in individual cells exposed to Gy level of reactor radiation. (author)

  12. Focal glomerulosclerosis in proviral and c-fms transgenic mice links Vpr expression to HIV-associated nephropathy

    International Nuclear Information System (INIS)

    Clinical and morphologic features of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN), such as proteinuria, sclerosing glomerulopathy, tubular degeneration, and interstitial disease, have been modeled in mice bearing an HIV proviral transgene rendered noninfectious through a deletion in gag/pol. Exploring the genetic basis of HIVAN, HIV transgenic mice bearing mutations in either or both of the accessory genes nef and vpr were created. Proteinuria and focal glomerulosclerosis (FGS) only developed in mice with an intact vpr gene. Transgenic mice bearing a simplified proviral DNA (encoding only Tat and Vpr) developed renal disease characterized by FGS in which Vpr protein was localized to glomerular and tubular epithelia by immunohistochemistry. The dual transgenic progeny of HIV[Tat/Vpr] mice bred to HIV[ΔVpr] proviral transgenic mice displayed a more severe nephropathy with no apparent increase in Vpr expression, implying that multiple viral genes contribute to HIVAN. However, the unique contribution of macrophage-specific Vpr expression in the development of glomerular disease was underscored by the induction of FGS in multiple murine lines bearing a c-fms/vpr transgene

  13. Mechanisms of DNA Repair in Mammalian Cells

    International Nuclear Information System (INIS)

    The authors examined DNA synthesis in cultured mammalian cells after irradiation with X-rays or ultraviolet light, using equilibrium density gradient and autoradiographic techniques. Unscheduled DNA synthesis (the synthesis of DNA by cells not in S phase of the cell cycle) occurs at doses of u.v. where survival is greater than 90% and at doses of X-rays where survival is of the order of 50%. At higher doses it was established that repair replication (insertion of precursors into parental strands of DNA ) occurs in these cells, and it is presumed that these two phenomena (unscheduled DNA synthesis and repair replication) are manifestations of the same repair process. During the time that these phenomena occur, very little degradation of DNA takes place, as measured by appearance of prelabelled components of DNA in the medium or in the acid soluble portion of the cell. This is in direct contrast to the situation in bacteria, in which extensive degradation of DNA occurs after irradiation, presumably as a result of enzymatic processes that remove many undamaged bases in addition to the ones injured by the irradiation. A small amount of radioactivity does appear in the acid soluble portion and in the media from prelabelled cells from both irradiated and control mammalian cell cultures. The amount in the medium from irradiated cultures is slightly, but significantly, greater than that from controls; thus there does appear to be a very low level of degradation of DNA in irradiated mammalian cells. These data indicate that the repair of DNA in mammalian cells does not involve the same steps as those that occur in bacteria. The results suggest instead that mammalian cells have a much more specific system for repair, which involves excision of only the damaged portion of the DNA. Possible mechanisms of repair of mammalian DNA are considered. (author)

  14. Mutagenesis and DNA repair in mammalian cells

    International Nuclear Information System (INIS)

    Two aspects of DNA damage and repair in mammalian cells were investigated. Using a lambda phage shuttle vector, a system was developed to study mutations arising in the DNA of mammalian cells. This system was used to determine the spectrum of mutations induced in cellular DNA by ultraviolet light. Also, the repair of base pair mismatches in DNA was studied by the development of a method to detect a DNA mismatch repair activity in extracts made from cultured human cells. In order to study mutations arising in mammalian cells, stable mouse L cell lines were established with multiple copies of lambda phage vector which contains the supF gene of E. coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient and yielded a negligible background of phage with mutations in the supF gene. From mouse cells exposed to 12 J/m2 of 254 nm ultraviolet (UV) light, 78,510 phage were rescued of which eight were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Py-C) sequences, and that the most frequent mutation at this site is a C to T transition

  15. Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

    OpenAIRE

    Firzlaff, J M; Diggelmann, H

    1984-01-01

    In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the tra...

  16. DNA repair in murine embryonic stem cells and differentiated cells

    International Nuclear Information System (INIS)

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

  17. Rearrangement of endogenous ecotropic proviral gene and interleukin-3 gene in radiation-induced myeloid leukemias of RFM/Un mice

    International Nuclear Information System (INIS)

    With the consideration that RFM/Un mice harbor on their chromosome 5 an ecotropic provirus locus for producing murine leukemia viruses (e-MuLV) and that interleukin-3 (IL-3) plays important roles in the growth and differentiation regulation of hematopoietic cells, particularly the myeloid lineages, they have examined radiation-induced myeloid leukemias of these mice for possible alterations in these two genes. Southern gel blot in combination with restriction enzyme analyses were performed with the use of an ecotropic env-specific sequence and an IL-3 cDNA clone as molecular probes. It was found that the transplantable Upton myeloid leukemia line has lost the chromosome 5 (germline) provirus and, instead, contains at least 4 copies of e-MuLV proviral sequences that have been acquired apparently by somatic re-integration. DNA preparations from 4 leukemic spleens of X-irradiated RFM/Un male mice, diagnosed histologically to have developed myeloid leukemias, were therefore examined; all four showed no loss of the germline e-MuLV copy and two of the four contained additional somatically re-integrated e-MuLV proviruses

  18. ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor.

    Science.gov (United States)

    Doria, Margherita; Tomaselli, Sara; Neri, Francesca; Ciafrè, Silvia Anna; Farace, Maria Giulia; Michienzi, Alessandro; Gallo, Angela

    2011-05-01

    The adenosine deaminases acting on RNA (ADAR) enzymes catalyse conversion of adenosine to inosine in dsRNA. A positive effect of ADAR1 on human immunodeficiency virus type 1 (HIV-1) replication has recently been reported. Here, we show that another ADAR enzyme, ADAR2, positively affects the replication process of HIV-1. We found that, analogously to ADAR1, ADAR2 enhances the release of progeny virions by an editing-dependent mechanism. However, differently from the ADAR1 enzyme, ADAR2 does not increase the infectious potential of the virus. Importantly, downregulation of ADAR2 in Jurkat cells significantly impairs viral replication. Therefore, ADAR2 shares some but not all proviral functions of ADAR1. These results suggest a novel role of ADAR2 as a viral regulator. PMID:21289159

  19. Sulfated fucan from marine alga inhibits HeLa cells infection by HTLV-1 free particles: semi-quantitative analysis

    Directory of Open Access Journals (Sweden)

    Maria T. V. Romanos

    2011-04-01

    Full Text Available A sulfated fucan from Laminaria abyssalis marine alga prevented the interaction of HTLV-1 particles, purified from the MT-2 cell line, with HeLa cells. The infection obtained using a concentrated virus suspension was detected only by amplification of the newly synthesized HTLV-1 proviral cDNA by the nested-polymerase chain reaction (PCR. The sulfated polysaccharide was not toxic to the cells at a concentration of 100 µg/mL and prevented infection by the viral particles when added to the cell monolayers. The proviral cDNA was only detected when the sulfated polysaccharide was added to the cells three hours post-infection, indicating that the inhibitory activity occurred in the initial stages of virus-cell interaction. Our results demonstrate, for the first time, the ability of a sulfated fucan from marine algae to inhibit virus transmission through free virus particles.

  20. Administration of a Toll-Like Receptor 9 Agonist Decreases the Proviral Reservoir in Virologically Suppressed HIV-Infected Patients

    OpenAIRE

    Winckelmann, Anni A.; Lærke V Munk-Petersen; Thomas A. Rasmussen; Jesper Melchjorsen; Thomas J Hjelholt; David Montefiori; Lars Østergaard; Søgaard, Ole S.; Martin Tolstrup

    2013-01-01

    Toll-like receptor (TLR) agonists can reactivate HIV from latently infected cells in vitro. We aimed to investigate the TLR-9 agonist, CPG 7909's in vivo effect on the proviral HIV reservoir and HIV-specific immunity. This was a post-hoc analysis of a double-blind randomized controlled vaccine trial. HIV-infected adults were randomized 1:1 to receive pneumococcal vaccines with or without 1 mg CPG 7909 as adjuvant at 0, 3 and 9 months. In patients on suppressive antiretroviral therapy we quant...

  1. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

  2. Cell-free DNA: Preanalytical variables.

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Aucamp, Janine; Pretorius, Piet J

    2015-10-23

    Since the discovery of cell-free DNA (cfDNA) in human blood, most studies have focused on diagnostic and prognostic uses of these markers for solid tumors. Except for some prenatal tests and BEAMing, cfDNA analysis has not yet been translated to clinical practice and routine application appears distant. This can be attributed to overlapping factors: (i) a lack of knowledge regarding the origin and function of cfDNA, (ii) insufficient molecular characterization, and (iii) the absence of an analytical consensus. In this review, we address the latter determinant and focus specifically on quantitative analysis of cfDNA. While the literature reports limited value for a single quantitative assessment, cfDNA kinetic assessment will be an essential component to qualitative characterization. In order to establish quantitative analysis for accurate kinetic assessments, process optimization and standardization are crucial. This report elucidates the most confounding variables of each preanalytic step that must be considered for optimal analysis. PMID:26341895

  3. DNA analysis of epithelial cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  4. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  5. Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

    OpenAIRE

    Ji Hyae Lim; Min Hyoung Kim; You Jung Han; Da Eun Lee; So Yeon Park; Jung Yeol Han; Moon Young Kim; Hyun Mee Ryu

    2013-01-01

    BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy....

  6. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces cell cycle synchronization in different human osteosarcoma cell lines. The UV pulse also has a destabilizing...

  7. Engineered cell-cell communication via DNA messaging

    Directory of Open Access Journals (Sweden)

    Ortiz Monica E

    2012-09-01

    Full Text Available Abstract Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.

  8. An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA

    OpenAIRE

    Deuring, Renate; Klotz, Günther; Doerfler, Walter

    1981-01-01

    On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into...

  9. DNA typing of epithelial cells after strangulation.

    Science.gov (United States)

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  10. Mitochondrial DNA mutations in single human blood cells.

    Science.gov (United States)

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  11. Proviral genomic sequence analysis of Chinese donkey leukocyte attenuated equine infectious anemia virus vaccine and its parental virus strain Liaoning

    Institute of Scientific and Technical Information of China (English)

    WANG; Liu(王柳); TONG; Guangzhi(童光志); LIU; Hongquan(刘红全); YANG; Zhibiao(杨志彪); QIU; Huaji(仇华吉); KONG; Xiangang(孔宪刚); WANG; Mei(王玫)

    2002-01-01

    Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated equine infectious anemia virus(DLA-EIAV), and peripheral blood lymphocytes(PBL) from a horse infected with the virulent EIAV strain Liaoning(EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV(DA-EIAV), respectively. Lots of variations occurred in long terminal repeat(LTR, consisting of U3, R, U5), ORF S2, and env regions between DLA-EIAV and EIAV L. The nucleotide sequence differences of the two viruses in U3, R, U5, ORF S2, and env are 13.2%, 7.5%, 5.1%, 3.9%, and 2.7%, respectively, and predicted amino acid sequence differences in env and S2 coding regions are 4.4% and 8.8%, respectively. Six conserved regions are characterized in Gp90. There is a cis-activating GATA motif in ENH of DLA-EIAV and EIAV L. Two N-linked glycosylation sites disappeared in DLA-EIAV Gp90 in comparison with that of EIAV L. A bHLH transcription factor binding consensus sequence was found in LTR of DLA-EIAV but not in EIAV L. Furthermore, there is a mutation in the stem of DLA-EIAV TAR resulting in formation of a uridine tuber. Further study is needed to uncover the relationship between sequence changes and their biological functions of DLA-EIAV and L.

  12. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect on the...

  13. 2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

    DEFF Research Database (Denmark)

    Ostrowski, S R; Ullum, H; Pedersen, B K;

    2005-01-01

    highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4...... expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected...... follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B...

  14. Detection of G-quadruplex DNA in mammalian cells

    NARCIS (Netherlands)

    Henderson, Alexander; Wu, Yuliang; Huang, Yu Chuan; Chavez, Elizabeth A.; Platt, Jesse; Johnson, F. Brad; Brosh, Robert M.; Sen, Dipankar; Lansdorp, Peter M.

    2014-01-01

    It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodi

  15. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60Co γ-rays, or to KUR radiation in the presence or absence of 10B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10B(n,α)7Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  16. Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

    OpenAIRE

    Mayanagi, Kouta; Kiyonari, Shinichi; Nishida, Hirokazu; Saito, Mihoko; Kohda, Daisuke; Ishino, Yoshizumi; Shirai, Tsuyoshi; Morikawa, Kosuke

    2011-01-01

    DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally ...

  17. Effect of gamma-irradiated DNA on DNA polymerase activity: a possible mechanism for cell killing

    International Nuclear Information System (INIS)

    A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. The mechanism for DNA polymerase inhibition was investigated. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme. The inhibition of DNA polymerase occurs prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation. As in vitro assay for DNA polymerase α and β in irradiated HeLa cells was developed. The activities of both polymerases decreased as the dose received by the cells increased. Both DNA polymerases were found to recover by 2 hr postirradiation. Since DNA repair capability is intimately connected with cell survival, the observed diminution in DNA polymerase activity, following low doses of radiation, could be highly significant

  18. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. METHODOLOGY/PRINCIPAL FINDINGS: A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively. CONCLUSIONS: Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA

  19. DNA conformational behavior and compaction in biomimetic systems: Toward better understanding of DNA packaging in cell.

    Science.gov (United States)

    Zinchenko, Anatoly

    2016-06-01

    In a living cell, long genomic DNA is strongly compacted and exists in the environment characterized by a dense macromolecular crowding, high concentrations of mono- and divalent cations, and confinement of ca. 10μm size surrounded by a phospholipid membrane. Experimental modelling of such complex biological system is challenging but important to understand spatiotemporal dynamics and functions of the DNA in cell. The accumulated knowledge about DNA condensation/compaction in conditions resembling those in the real cell can be eventually used to design and construct partly functional "artificial cells" having potential applications in drug delivery systems, gene therapy, and production of synthetic cells. In this review, I would like to overview the past progress in our understanding of the DNA conformational behavior and, in particular, DNA condensation/compaction phenomenon and its relation to the DNA biological activity. This understanding was gained by designing relevant experimental models mimicking DNA behavior in the environment of living cell. Starting with a brief summary of classic experimental systems to study DNA condensation/compaction, in later parts, I highlight recent experimental methodologies to address the effects of macromolecular crowding and nanoscale and microscale confinements on DNA conformation dynamics. All the studies are discussed in the light of their relevance to DNA behavior in living cells, and future prospects of the field are outlined. PMID:26976700

  20. DNA Charge Transport within the Cell

    OpenAIRE

    Grodick, Michael A.; Muren, Natalie B.; Barton, Jacqueline K.

    2015-01-01

    The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor th...

  1. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

    Science.gov (United States)

    Znidar, Katarina; Bosnjak, Masa; Cemazar, Maja; Heller, Loree C

    2016-01-01

    In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo. PMID:27271988

  2. DNA damage and repair in human cells exposed to sunlight

    International Nuclear Information System (INIS)

    Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (>320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful. (author)

  3. Cosegregation of cell wall and DNA in Bacillus subtilis.

    OpenAIRE

    Schlaeppi, J M; Karamata, D

    1982-01-01

    Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respec...

  4. DNA nanotechnology from the test tube to the cell

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  5. Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

    International Nuclear Information System (INIS)

    Three techniques single cell gel electrophoresis (SCGE), alkaline elution of DNA, and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cell exposed to hydrogen peroxide in concentration range from 10 μmol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 μmol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 μmol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyridine-DNA glycosylase. (author)

  6. Cell-free DNA: Comparison of Technologies.

    Science.gov (United States)

    Dar, Pe'er; Shani, Hagit; Evans, Mark I

    2016-06-01

    Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies. PMID:27235906

  7. Mitochondrial DNA determines androgen dependence in prostate cancer cell lines

    OpenAIRE

    Higuchi, M; Kudo, T; Suzuki, S.; Evans, TT; Sasaki, R.; Wada, Y; Shirakawa, T.; Sawyer, JR; Gotoh, A

    2006-01-01

    Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is verycommon in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstra...

  8. New, small circular DNA in transfected mammalian cells.

    OpenAIRE

    Wiberg, F C; Sunnerhagen, P; Bjursell, G

    1986-01-01

    Circular DNA isolated by the Hirt procedure from transfected mammalian cells was examined by electron microscopy. Typically, the number of small (1- to 5-kilobase) DNA circles increased about fivefold even though DNA of larger size classes (5 to 15 kilobases) has been transferred. In one case, where extensive rearrangement of the transferred DNA was observed, the rearrangement products were cloned and analyzed. In most cases, however, no rearrangement could be detected, but the amount of smal...

  9. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Science.gov (United States)

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  10. DNA content analysis of insect cell lines by flow cytometry

    OpenAIRE

    Léry, Xavier; Charpentier, Guy; Belloncik, Serge

    1999-01-01

    The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cel...

  11. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    OpenAIRE

    Philip Burnham; Min Seong Kim; Sean Agbor-Enoh; Helen Luikart; Hannah A. Valantine; Kiran K Khush; Iwijn De Vlaminck

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in...

  12. Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

    OpenAIRE

    Hirotake Imanishi; Keisuke Hattori; Reiko Wada; Kaori Ishikawa; Sayaka Fukuda; Keizo Takenaga; Kazuto Nakada; Jun-ichi Hayashi

    2011-01-01

    Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously e...

  13. High-efficiency transformation of mammalian cells by plasmid DNA.

    OpenAIRE

    Chen, C.; Okayama, H

    1987-01-01

    We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cell...

  14. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  15. Comparative study of the free radical and DNA break accumulation under gamma-irradiation of DNA solutions and cells

    International Nuclear Information System (INIS)

    The linear dependence between parameters of DNA radiation dmage is disclosed in the given paper in determining the concentration of free radicals and the number of DNA breaks in the same samples of irradiated frozen DNA solutions, cells and tissues

  16. A two-dimensional DNA lattice implanted polymer solar cell

    International Nuclear Information System (INIS)

    A double crossover tile based artificial two-dimensional (2D) DNA lattice was fabricated and the dry-wet method was introduced to recover an original DNA lattice structure in order to deposit DNA lattices safely on the organic layer without damaging the layer. The DNA lattice was then employed as an electron blocking layer in a polymer solar cell causing an increase of about 10% up to 160% in the power conversion efficiency. Consequently, the resulting solar cell which had an artificial 2D DNA blocking layer showed a significant enhancement in power conversion efficiency compared to conventional polymer solar cells. It should be clear that the artificial DNA nanostructure holds unique physical properties that are extremely attractive for various energy-related and photonic applications.

  17. Prevention of DNA re-replication in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Lan N. Truong; Xiaohua Wu

    2011-01-01

    DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability.

  18. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

  19. DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells

    International Nuclear Information System (INIS)

    When mammalian cells are lysed in 2% sodium dodecyl sulphate detergent followed by addition of an equal volume of 0.12 M potassium chloride, a precipitate forms that can be collected by low-speed centrifugation. This precipitate contains the cell protein and nucleic acid in close association with protein. In the absence of DNA damage, most of the DNA precipitates, but when DNA strand breaks are created by exposing cells to ionizing radiation or toxic chemicals, DNA is released from the protein and remains in the supernatant after centrifugation. The proportion of DNA remaining in the supernatant is thus a measure of the amount of DNA damage. This assay is characterized in terms of optimum cell number and pH and dose-response curves for DNA damage and cell survival following ionizing radiation, MNNG, BCNU, and VP-16 are shown. Sensitivity, simplicity, speed, and large sample handling capacity should allow wide application of this new assay to a variety of questions concerning DNA damage and repair

  20. Quantitative analysis of cell-free DNA in ovarian cancer

    OpenAIRE

    Shao, Xuefeng; He, Yan; Ji, Min; Chen, Xiaofang; Qi, Jing; SHI, Wei; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ova...

  1. Detection of hepatitis B virus DNA in mononuclear blood cells.

    OpenAIRE

    Pontisso, P; Poon, M C; Tiollais, P.; Brechot, C

    1984-01-01

    The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and on...

  2. Cell cycle control after DNA damage: arrest, recovery and adaptation

    International Nuclear Information System (INIS)

    DNA damage triggers surveillance mechanisms, the DNA checkpoints, that control the genome integrity. The DNA checkpoints induce several responses, either cellular or transcriptional, that favor DNA repair. In particular, activation of the DNA checkpoints inhibits cell cycle progression in all phases, depending on the stage when lesions occur. These arrests are generally transient and cells ultimately reenter the cell division cycle whether lesions have been repaired (this process is termed 'recovery') or have proved un-repairable (this option is called 'adaptation'). The mechanisms controlling cell cycle arrests, recovery and adaptation are largely conserved among eukaryotes, and much information is now available for the yeast Saccharomyces cerevisiae, that is used as a model organism in these studies. (author)

  3. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  4. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

    Science.gov (United States)

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A; Kumar, Sheetal; Kalab, Petr

    2016-04-15

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  5. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    Directory of Open Access Journals (Sweden)

    Mark R. Openshaw

    2016-02-01

    Full Text Available Gestational trophoblastic neoplasia (GTN represents a group of diseases characterized by production of human chorionic gonadotropin (hCG. Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA (from 9% to 53% of total cfDNA in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis.

  6. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund;

    2015-01-01

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are...... diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In...

  7. Cell-cell contact viral transfer contributes to HIV infection and persistence in astrocytes

    OpenAIRE

    Luo, Xiaoyu; He, Johnny J.

    2014-01-01

    Astrocytes are the most abundant cells in the central nervous system and play important roles in HIV/neuroAIDS. Detection of HIV proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV infection albeit in a restricted manner. We as well as others have shown that cell-free HIV is capable of entering CD4− astrocytes through human mannose receptor-mediated endocytosis. In this study, we to...

  8. Mitochondrial DNA sequence variation in single cells from leukemia patients

    OpenAIRE

    Yao, Yong-Gang; Ogasawara, Yoji; Kajigaya, Sachiko; Molldrem, Jeffrey J.; Falcão, Roberto P; Pintão, Maria-Carolina; McCoy, J. Philip; Rizzatti, Edgar Gil; Young, Neal S

    2007-01-01

    A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a popu...

  9. DNA profiles generated from minute amounts of single cells

    OpenAIRE

    Wenäll, Lovisa

    2011-01-01

    The genetic code in our cells is built up by deoxyribonucleic acid (DNA) with a sequence that is individual and unique to each person. A cell’s origin can be decided by comparing an established DNA profile with a known profile. The most publicly known application is in the forensic field and its use for identification and for establishing a connection between perpetrators and victims or crime scenes. DNA profiling is also commonly used for kinship investigations. The information embedded in t...

  10. Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations.

    OpenAIRE

    Pane, F; Buttò, S; Gobbo, M L; Franco, M; Butteroni, C; Pastore, L; G. Maiorano; Foggia, M; Cataldo, P T; Guarino, A

    1995-01-01

    We used a colorimetric polymerase chain reaction (PCR)-based assay in kit form to detect directly human immunodeficiency virus type 1 (HIV-1) proviral gag sequences in peripheral blood cells from 68 healthy blood donors, 51 subjects at risk for HIV infection, 122 patients with HIV-1 infection, 11 patients with indeterminate Western blot (immunoblot) results, 4 blood donors HIV-1 positive by enzyme immunoassay, and 13 children born to HIV-1-seropositive mothers. The results obtained in the blo...

  11. Analysis of epigenetic modifications of DNA in human cells

    DEFF Research Database (Denmark)

    Kristensen, Lasse Sommer; Treppendahl, Marianne Bach; Grønbæk, Kirsten

    2013-01-01

    Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found...

  12. Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site.

    Science.gov (United States)

    Senigl, Filip; Auxt, Miroslav; Hejnar, Jirí

    2012-07-01

    The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a(-/-) Dnmt3b(-/-) cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression. PMID:22379139

  13. DNA Charge Transport: from Chemical Principles to the Cell.

    Science.gov (United States)

    Arnold, Anna R; Grodick, Michael A; Barton, Jacqueline K

    2016-01-21

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

  14. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  15. DNA breaks early in replication in B cell cancers

    Science.gov (United States)

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  16. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  17. Decreased serum cell-free DNA levels in rheumatoid arthritis

    OpenAIRE

    Dunaeva, Marina; Buddingh’, Bastiaan C.; René E M Toes; Luime, Jolanda J.; Lubberts, Erik; Pruijn, Ger J. M.

    2015-01-01

    Purpose Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods cfDNA was extracted from sera of patients with early and established RA, relapsing-remitt...

  18. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  19. UV stimulation of DNA-mediated transformation of human cells

    International Nuclear Information System (INIS)

    Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells, which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome

  20. Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells.

    Directory of Open Access Journals (Sweden)

    Andreas Luch

    Full Text Available OBJECTIVE: Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. METHODOLOGY/PRINCIPAL FINDINGS: The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding and XPC (xeroderma pigmentosum group C was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. CONCLUSIONS/SIGNIFICANCE: A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks.

  1. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

    OpenAIRE

    Barrett, A. N.; Zimmermann, B. G.; Wang, D.; Holloway, A.; Chitty, L S

    2011-01-01

    Objective: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing ce...

  2. Somatic cell genetics approach to dissecting mammalian DNA repair

    International Nuclear Information System (INIS)

    This review article examines the application of the methods and concepts of somatic cell genetics to the study of DNA repair. The first steps of this approach involve classical procedures of mutant isolation, complementation analysis, and mapping of genes using hybrid cells. Subsequent steps utilize the techniques of DNA-mediated gene transfer and methodologies of the recombinant DNA field. Several human repair genes have been cloned, but they have not been used to overproduce proteins thus far. This article highlights the more important developments and attempts to review in detail all of the isolated mutant cell lines that may be altered in the repair processes. Faster methods of gene cloning are greatly needed because the procedures for making secondary transformants from total genomic DNA are tedious

  3. Murine thymic lymphomas after infection with a B-ecotropic murine leukemia virus and/or X-irradiation: proviral organization and RNA expression

    International Nuclear Information System (INIS)

    The role of retroviruses in murine radioleukemogenesis was reinvestigated using a protocol associating the injection of a non-pathogenic retrovirus (T1223/B virus) and a subleukemogenic dose of X-radiation (2 x 1.75 Gy). Using the Southern blotting technique the authors studied MuLV proviral organization and RNA expression in thymic lymphomas induced by the combined effect of virus and irradiation or irradiation alone. A recombinant provirus was detected in the chromosomal DNA of every tumor induced by associating virus and radiation whereas it was not always found in radio-induced tumors. In every instance, the provirus was not integrated at a common site. No relationship was observed between viral RNA expression and tumor induction. Trisomy 15 was observed in all metaphases irrespective of the protocol of tumor induction. The G-banding technique revealed an extra-band in several thymic lymphomas induced by irradiation and T1223/B virus injection. (author)

  4. Evaluation of eukaryotic cultured cells as a model to study extracellular DNA / D.L. Peters

    OpenAIRE

    Peters, Dimetrie Leslie

    2011-01-01

    The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis....

  5. A novel cell permeable DNA replication and repair marker

    OpenAIRE

    Herce, Henry D.; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M. Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA bindin...

  6. Acetylation regulates DNA repair mechanisms in human cells.

    Science.gov (United States)

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  7. Roles of host cell factors in circularization of retroviral DNA

    International Nuclear Information System (INIS)

    Early during retroviral infection, a fraction of the linear reverse-transcribed viral DNA genomes become circularized by cellular enzymes, thereby inactivating the genomes for further replication. Prominent circular DNA forms include 2-long-terminal repeat (LTR) circles, made by DNA end joining, and 1-LTR circles, produced in part by homologous recombination. These reactions provide a convenient paradigm for analyzing the cellular machinery involved in DNA end joining in vertebrate cells. In previous studies, we found that inactivating components of the nonhomologous DNA end-joining (NHEJ) pathway--specifically Ku, ligase 4, or XRCC4--blocked formation of 2-LTR circles. Here we report that inactivating another NHEJ component, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), had at most modest effects on 2-LTR circle formation, providing informative parallels with other end-joining reactions. We also analyzed cells mutant in components of the RAD50/MRE11/NBS1 nuclease and found a decrease in the relative amount of 1-LTR circles, opposite to the effects of NHEJ mutants. In MRE11-mutant cells, a MRE11 gene mutant in the nuclease catalytic site failed to restore 1-LTR circle formation, supporting a model for the role of MRE11 in 1-LTR circle formation. None of the cellular mutations showed a strong effect on normal integration, consistent with the idea that the cellular pathways leading to circularization are not involved in productive integration

  8. Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

    OpenAIRE

    Montigny, William J.; Houchens, Christopher R.; Illenye, Sharon; Gilbert, Jonathan; Coonrod, Emily; Chang, Young-Chae; Nicholas H. Heintz

    2001-01-01

    Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 1...

  9. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  10. Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline.

    Science.gov (United States)

    Chalvet, F; Teysset, L; Terzian, C; Prud'homme, N; Santamaria, P; Bucheton, A; Pélisson, A

    1999-05-01

    Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed. PMID:10228177

  11. Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

    Science.gov (United States)

    De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

    2016-07-27

    The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. PMID:27298260

  12. Characterization of the cell-free DNA released by cultured cancer cells.

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Wentzel, Johannes F; Aucamp, Janine; van Dyk, Etresia; du Plessis, Lissinda; Pretorius, Piet J

    2016-01-01

    The most prominent factor that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in the blood of healthy and diseased individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer patients is not only representative of tumor derived DNA, but also of DNA released by healthy cells under different conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant, but likely includes non-mutated chromosomal DNA fragments. This may cause false positive/negative results. Although many have acknowledged that this is a major problem, few have addressed it. We propose that many of the current stumbling blocks encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Results suggest that the occurrence of cfDNA is not a consequence of apoptosis or necrosis, but primarily a result of actively secreted DNA, perhaps in association with a protein complex. This study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA. PMID:26529550

  13. Chromatin factors affecting DNA repair in mammalian cell nuclei

    International Nuclear Information System (INIS)

    We are investigating chromatin factors that participate in the incision step of DNA repair in eukaryotic cells. Localization of repair activity within nuclei, the stability and extractability of activity, the specificity for recognizing damage in chromatin or purified DNA as substrates are of interest in this investigation of human cells, CHO cells, and their radiation sensitive mutants. We have developed procedures that provide nuclei in which their DNA behaves as a collection of circular molecules. The integrity of the DNA in human nuclei can be maintained during incubation in appropriate buffers for as long as 60 minutes. When cells or nuclei are exposed to uv light prior to incubation, incisions presumably associated with DNA repair can be demonstrated. Incision activity is stable to prior extraction of nuclei with 0.6 M NaCl, which removes many nonhistone proteins. Our studies are consistent with an hypothesis that factors responsible for initiating DNA repair are localized in the nuclear matrix. 18 references, 3 figures

  14. Quantitative analysis of cell-free DNA in ovarian cancer

    Science.gov (United States)

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  15. Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat.

    Science.gov (United States)

    Jadlowsky, Julie K; Wong, Julian Y; Graham, Amy C; Dobrowolski, Curtis; Devor, Renee L; Adams, Mark D; Fujinaga, Koh; Karn, Jonathan

    2014-06-01

    The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses. PMID:24636995

  16. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  17. Scintillometric determination of DNA repair in human cell lines

    International Nuclear Information System (INIS)

    The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [3H]thymidine, the radioactivity incorporated into DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (Csub(HU), Tsub(HU)). The ratios Tsub(HU)/Csub(HU) and Tsub(HU)/T:Csub(HU)/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation. (orig./AJ)

  18. New Rapid Method of DNA Isolation from Milk Somatic Cells.

    Science.gov (United States)

    Pokorska, Joanna; Kułaj, Dominika; Dusza, Magdalena; Żychlińska-Buczek, Justyna; Makulska, Joanna

    2016-04-01

    Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses. PMID:26913552

  19. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  20. Toxicity DNA damage and inhibition of DNA repair synthesis in human melanoma cells by concentrated sunlight

    International Nuclear Information System (INIS)

    A water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line, DNA strand breaks and DNA protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers and DNA interstrand crosslinking could not be detected. The solar fluence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D0 value and then declining; but semiconservative DNA synthesis remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis. (author)

  1. PREPARATION AND PURIFICATION OF DNA FROM BACTERIAL CELLS; CHARACTERIZATION OF PLASMID DNA

    Directory of Open Access Journals (Sweden)

    M Praveen, G Adarsh, T Ramesh, M Ramesh*

    2013-01-01

    Full Text Available The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. With the advancement in genetic engineering, foreign genes of industrial applications can be inserted into cloning vector for mass production in various host cells. Escherichia coli is an extremely important model organism in modern biological engineering, the suitable growth media is essential for the optimal expression of the genes in E. coli. The present study aims at isolation and purification of genomic DNA from E. coli, the characterization of pBR322 plasmid DNA. Bacterial culture conditions were optimized in shake – flask cultures based on optimal temperature, inoculum size and medium composition. Solutions and methods are disclosed for the effective, simple isolation of DNA from bacterial cells. High bioprocess recovery and product quality were primarily associated with the complete removal of total cellular RNA impurity. The process was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW RNA and other impurities were removed by selective precipitation using calcium chloride at an optimal concentration.The optimal conditions for the growth of Escherichia coli were shown maximum absorbance as 7.5 at 370C temperature, 1% inoculum size using TB medium composition. The purified genomic DNA had concentration as 73.5 µg/ml and purity 1.8. The 0.5M CaCl2 was optimal concentration for removal of RNA. The plasmid DNA pBR322 was confirmed by comparing the band to 4.36 Kb, purity of plasmid was 1.85 and it contains 96.8% of super coiled DNA. The contaminants like chromosomal DNA, RNA, host cell proteins and mycoplasma were absent in the plasmid DNA.

  2. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas

    Directory of Open Access Journals (Sweden)

    Roth Sanford I

    2007-10-01

    Full Text Available Abstract Background The potential pathogenetic significance of mitochondrial DNA (mtDNA mutations in tumorigenesis is controversial. We hypothesized that benign tumorigenesis of a slowly replicating tissue like the human parathyroid might constitute an especially fertile ground on which a selective advantage conferred by mtDNA mutation could be manifested and might contribute to the oxyphilic phenotype observed in a subset of parathyroid tumors. Methods We sought acquired mitochondrial DNA mutations by sequencing the entire 16.6 kb mitochondrial genome of each of thirty sporadic parathyroid adenomas (18 chief cell and 12 oxyphil cell, eight independent, polyclonal, parathyroid primary chief cell hyperplasias plus corresponding normal control samples, five normal parathyroid glands, and one normal thyroid gland. Results Twenty-seven somatic mutations were identified in 15 of 30 (9 of 12 oxyphil adenomas, 6 of 18 chief cell parathyroid adenomas studied. No somatic mutations were observed in the hyperplastic parathyroid glands. Conclusion Features of the somatic mutations suggest that they may confer a selective advantage and contribute to the molecular pathogenesis of parathyroid adenomas. Importantly, the statistically significant differences in mutation prevalence in oxyphil vs. chief cell adenomas also suggest that mtDNA mutations may contribute to the oxyphil phenotype.

  3. Study of DNA uptake locations in single E. coli cells

    Science.gov (United States)

    Xu, C. Shan; Meadow Anderson, L.; Yang, Haw

    2006-03-01

    Artificial gene transfer of bacteria, such as E. coli, has become the main stream technique in genetic engineering and molecular cell biology studies. In spite of the great improvements in transformation efficiency, some fundamental questions remained to be answered. For instance, what are the DNA uptake channels and how do they form and function under external stimuli? Furthermore, where are these channels located on the cell membrane? Here we report a study aimed at DNA uptake locations in the two widely used gene transformation techniques: electroporation and heat shock. A direct visualization of the settling location of single DNA molecules inside individual E. coli cells was obtained by fluorescence imaging and spectroscopy. Electroporation and heat shock exhibit two distinct characteristics of DNA uptake locations. A preferential distribution toward cell poles during electroporation is consistent with earlier experiments and previously proposed models. However, the result from heat shock is unanticipated in which the majority of DNA enters the cell near the center. Such observation suggests that uptake channels form preferentially where newly-synthesized membrane is located under cation and low temperature treatment

  4. Human Immunodeficiency Virus Type 1 DNA Sequences Genetically Damaged by Hypermutation Are Often Abundant in Patient Peripheral Blood Mononuclear Cells and May Be Generated during Near-Simultaneous Infection and Activation of CD4+ T Cells

    OpenAIRE

    Janini, Mario; Rogers, Melissa; Birx, Deborah R.; McCutchan, Francine E.

    2001-01-01

    G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 proteas...

  5. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    OpenAIRE

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J; Baljeet Kaur; Naveed Sarwar; Michael J Seckl; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients wit...

  6. Controls to validate plasma samples for cell free DNA quantification.

    Science.gov (United States)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund; Brandslund, Ivan; Jakobsen, Anders

    2015-06-15

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology. PMID:25896958

  7. Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

    OpenAIRE

    S. Kato; Anderson, R. A; Camerini-Otero, R. D.

    1986-01-01

    We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail ...

  8. Cadmium Induced Cell Apoptosis, DNA Damage, Decreased DNA Repair Capacity, and Genomic Instability during Malignant Transformation of Human Bronchial Epithelial Cells

    OpenAIRE

    Zhou, Zhiheng; Wang, Caixia; Liu, Haibai; Huang, Qinhai; Wang, Min; Lei, Yixiong

    2013-01-01

    Cadmium and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood. Our study was designed to elucidate the mechanisms of DNA damage in cadmium-induced malignant transformation of human bronchial epithelial cells. We analyzed cell cycle, apoptosis, DNA damage, gene expression, genomic instability, and the sequence of exons in DNA repair genes in several kinds of cells. These cells consisted of untreated control cells, cells...

  9. Single Cell Gel Electrophoresis in DNA Damage Detection (Comet Assay)

    OpenAIRE

    Aysen Durmaz; Nurten Dikmen; Cumhur Gunduz

    2010-01-01

    “Single cell gel electrophoresis (SCGE)”, also called “Comet Assay”, is a sensitive, reliable and rapid technique for quantifying and analyzing DNA damage in individual cells. The comet assay is widely used in living cells, researches and the applications on comet assay is becoming broader day by day. To date, the comet assay has been used for a variety of applications, including genotoxic and cytotoxic agent analyses, environmental toxicology, cancer research, and radiati...

  10. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    OpenAIRE

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; DE GIORGI, VINCENZO; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the pres...

  11. Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells

    International Nuclear Information System (INIS)

    We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T). The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells. This permeabilization can be reversed by diluting the cells in growth medium. Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation. Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells. A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells. Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type. It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion. It has a molecular weight (MW) in the range 20-30 000 D. The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible

  12. DNA precursor compartmentation in mammalian cells: metabolic and antimetabolic studies of nuclear and mitochondrial DNA synthesis

    International Nuclear Information System (INIS)

    HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay of dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochrondrial rNTP pools were also found to expand in response to these antimetabolites. Mouse L-cells were used to determine the relative contributions of an exogenously supplied precursor to nuclear and mitochrondrial DNA replication. Cells were labeled to near steady state specific activities with 32P-orthophosphate and subsequently labeled with [3H]uridine, a general pyrimidine precursor, in the continuing presence of 32P. Deoxyribonucleoside monophosphates derived from these DNAs were separated by HPLC and the 3H/32P ratio in each pyrimidine determined. The dCMP residues in mitochondrial DNA (mtDNA) were found to be derived exclusively from the exogenous supplied uridine. The dTMP residues from nuclear and mtDNA and the dCMP residues from nuclear DNA were seen to be synthesized partly from exogenous sources and partly from other sources, presumably de novo pyrimidine synthesis

  13. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  14. DNA mismatch repair efficiency and fidelity are elevated during DNA synthesis in human cells

    International Nuclear Information System (INIS)

    DNA mismatch repair (MMR) within human cells is hypothesized to occur primarily at the replication fork. However, experimental models measuring MMR activity at specific phases of the cell cycle and during genomic DNA synthesis are lacking. We have investigated MMR activity within the nuclear environment of HeLa cells after enriching for G1, S and G2/M phase of the cell cycle by centrifugal elutriation. This approach preserves physiologically normal MMR activity in cell populations subdivided into different phases of the cell cycle. Here we have shown that nuclear protein concentration of hMutSα and hMutLα increases as cells progress into S phase during routine cell culture. MMR activity, as measured by both in vitro and in vivo approaches, increases during S phase to the highest extent within normally growing cells. Both fidelity and activity of MMR are highest on actively replicating templates within intact cells during S phase. The MMR pathway however, is also active at lower levels at other phases of the cell cycle, and on nonreplicating templates

  15. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    We have reported that mitochondrial DNA-depleted ρ0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ0 cells. These results support the mitochondrial theory of aging, and suggest that ρ0 cells could serve as an in vitro model for aged cells

  16. Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells

    Science.gov (United States)

    Zhou, Yang; Hu, Zhengqing

    2016-01-01

    The DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) causes genomic demethylation to regulate gene expression. However, it remains unclear whether 5-aza affects gene expression and cell fate determination of stem cells. In this study, 5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination. PMID:27536218

  17. The use of recombinant DNA plasmids for the determination of DNA-repair and recombination in cultured mammalian cells.

    OpenAIRE

    Cox, R.; Masson, W. K.; Debenham, P G; Webb, M. B.

    1984-01-01

    Using the recombinant plasmid pSV2gpt and DNA transfer techniques, cell mediated DNA ligation and recombination of plasmid DNA have been demonstrated in four human cell lines. Data suggesting the involvement of a possible defect in the cellular equilibrium between ligation and exonuclease digestion of double strand DNA scissions in an ataxia-telangiectasia (A-T) cell line is discussed. The same A-T line was grossly proficient in DNA recombination but it will be necessary to distinguish betwee...

  18. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  19. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Science.gov (United States)

    Reinson, Tormi; Henno, Liisi; Toots, Mart; Ustav, Mart; Ustav, Mart

    2015-01-01

    Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research. PMID:26132923

  20. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Directory of Open Access Journals (Sweden)

    Tormi Reinson

    Full Text Available Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

  1. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2016-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects. PMID:26703663

  2. Does 'Immortal DNA strand' exist in 'immortal' stem cells?

    Institute of Scientific and Technical Information of China (English)

    Linheng Li

    2007-01-01

    @@ Stem cells function to generate differentiated cells,and,at the same time,are maintained as‘immortal'cells through self-renewal.Accumulated evidence indicates that adult stem cells may be the most common precursor for cancers in adult mammalian tissue[1],and this concept is gaining increasing support[2,3].Meanwhile,this also raises the question of how stem cells,which have a much longer lifespan compared to other progenitor and mature ceils,can avoid accumulating mutations from DNA replica-tion errors.

  3. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

    Directory of Open Access Journals (Sweden)

    Angela N Barrett

    Full Text Available OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA. Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3EDTA tubes can be used. Prolonged transfer times in K(3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

  4. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng;

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...

  5. Relationship between DNA damage, rejoining and cell killing by radiation in mammalian cells

    International Nuclear Information System (INIS)

    The prevailing hypothesis on the mechanism of radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. In this review we present new data and summarize the role of the DNA double-strand breaks (dsb) induced by ionizing radiation and DNA dsb rejoining as determinants of cellular radiosensitivity. When cells were irradiated at high dose-rate, two molecular end-points were identified which often correlated with radiosensitivity: (1) the apparent number of DNA dsb induced per Gy per DNA unit and (2) the half-time of the fast component of the DNA dsb rejoining kinetics. These two molecular determinants, not mutually exclusive, may be linked through a common factor such as the conformation of DNA

  6. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  7. The DNA damage-induced cell death response: a roadmap to kill cancer cells.

    Science.gov (United States)

    Matt, Sonja; Hofmann, Thomas G

    2016-08-01

    Upon massive DNA damage cells fail to undergo productive DNA repair and trigger the cell death response. Resistance to cell death is linked to cellular transformation and carcinogenesis as well as radio- and chemoresistance, making the underlying signaling pathways a promising target for therapeutic intervention. Diverse DNA damage-induced cell death pathways are operative in mammalian cells and finally culminate in the induction of programmed cell death via activation of apoptosis or necroptosis. These signaling routes affect nuclear, mitochondria- and plasma membrane-associated key molecules to activate the apoptotic or necroptotic response. In this review, we highlight the main signaling pathways, molecular players and mechanisms guiding the DNA damage-induced cell death response. PMID:26791483

  8. Significance of DNA quantification in testicular germ cell tumors.

    Science.gov (United States)

    Codesal, J; Paniagua, R; Regadera, J; Fachal, C; Nistal, M

    1991-01-01

    A cytophotometric quantification of DNA in tumor cells was performed in histological sections of orchidectomy specimens from 36 men with testicular germ cell tumors (TGCT), 7 of them showing more than one tumor type. Among the variants of seminoma (classic and spermatocytic) the lowest DNA content were in spermatocytic seminoma. With respect to non-seminomatous tumors (yolk sac tumor, embryonal carcinoma, teratoma, and choriocarcinoma), choriocarcinomas showed the highest DNA content, and the lowest value was found in teratomas. No significant differences were found between the average DNA content of seminomas (all types) and non-seminomatous tumors (all types). Both embryonal carcinoma and yolk sac tumor showed similar DNA content when they were the sole tumor and when they were found associated with other tumors. In this study, except for the 4 cases of teratoma and the case of spermatocytic seminoma, all TGCT examined did not show modal values of DNA content in the diploid range. Such an elevated frequency of aneuploidism in these tumors may be helpful for their diagnosis. PMID:1666273

  9. Proviral integrations and expression of endogenous Avian leucosis virus during long term selection for high and low body weight in two chicken lines

    Directory of Open Access Journals (Sweden)

    Bornold Lina

    2009-07-01

    Full Text Available Abstract Background Long-term selection (> 45 generations for low or high juvenile body weight from a common founder population of White Plymouth Rock chickens has generated two extremely divergent lines, the LWS and HWS lines. In addition to a > 9-fold difference between lines for the selected trait, large behavioural and metabolic differences between the two lines evolved during the course of the selection. We recently compared gene expression in brain tissue from birds representing these lines using a global cDNA array analysis and the results showed multiple but small expression differences in protein coding genes. The main differentially expressed transcripts were endogenous retroviral sequences identified as avian leucosis virus subgroup-E (ALVE. Results In this work we confirm the differential ALVE expression and analysed expression and number of proviral integrations in the two parental lines as well as in F9 individuals from an advanced intercross of the lines. Correlation analysis between expression, proviral integrations and body weight showed that high ALVE levels in the LWS line were inherited and that more ALVE integrations were detected in LWS than HWS birds. Conclusion We conclude that only a few of the integrations contribute to the high expression levels seen in the LWS line and that high ALVE expression was significantly correlated with lower body weights for the females but not males. The conserved correlation between high expression and low body weight in females after 9 generations of intercrosses, indicated that ALVE loci conferring high expression directly affects growth or are very closely linked to loci regulating growth.

  10. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Ganna Bilousova

    2005-12-01

    Full Text Available Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.

  11. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  12. Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

    Science.gov (United States)

    Hofer, Michal; Falk, Martin; Komůrková, Denisa; Falková, Iva; Bačíková, Alena; Klejdus, Bořivoj; Pagáčová, Eva; Štefančíková, Lenka; Weiterová, Lenka; Angelis, Karel J; Kozubek, Stanislav; Dušek, Ladislav; Galbavý, Štefan

    2016-04-14

    Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration. PMID:26978566

  13. Strategies for Implementing Cell-Free DNA Testing.

    Science.gov (United States)

    Cuckle, Howard

    2016-06-01

    Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. PMID:27235907

  14. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

    Science.gov (United States)

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A; Khush, Kiran K; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  15. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure

    Science.gov (United States)

    Xu, Qiongqiong; Luo, Juntao; Shi, Yingdi; Li, Xiaoxia; Yan, Xiaonan; Zhang, Junfang

    2016-01-01

    DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure. PMID:27494266

  16. Blood cell mitochondrial DNA content and premature ovarian aging.

    Directory of Open Access Journals (Sweden)

    Marco Bonomi

    Full Text Available Primary ovarian insufficiency (POI is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA content in a group of women undergoing ovarian hyperstimulation (OH, and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF and 42 poor responders (PR to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001 in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

  17. Mutations in Ovis aries TMEM154 are associated with lower small ruminant lentivirus proviral concentration in one sheep flock.

    Science.gov (United States)

    Alshanbari, F A; Mousel, M R; Reynolds, J O; Herrmann-Hoesing, L M; Highland, M A; Lewis, G S; White, S N

    2014-08-01

    Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection. PMID:24934128

  18. Multiparametric analysis of cell-free DNA in melanoma patients.

    Directory of Open Access Journals (Sweden)

    Francesca Salvianti

    Full Text Available Cell-free DNA in blood (cfDNA represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF(V600E mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC. To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model was weak/satisfactory ranging between 0.64 (BRAF(V600E to 0.85 (total cfDNA. A good overall predictive capability for the final logistic model was found with an AUC of 0.95. The highest predictive capability was given by total cfDNA (AUC:0.86 followed by integrity index 180/67 (AUC:0.90 and methylated RASSF1A (AUC:0.89.An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A could improve the diagnostic performance in melanoma.

  19. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

    International Nuclear Information System (INIS)

    SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

  20. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    International Nuclear Information System (INIS)

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair

  1. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  2. DNA profiling and characterization of animal cell lines.

    Science.gov (United States)

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

  3. Cell cycle control of DNA joint molecule resolution.

    Science.gov (United States)

    Wild, Philipp; Matos, Joao

    2016-06-01

    The establishment of stable interactions between chromosomes underpins vital cellular processes such as recombinational DNA repair and bipolar chromosome segregation. On the other hand, timely disengagement of persistent connections is necessary to assure efficient partitioning of the replicated genome prior to cell division. Whereas great progress has been made in defining how cohesin-mediated chromosomal interactions are disengaged as cells prepare to undergo chromosome segregation, little is known about the metabolism of DNA joint molecules (JMs), generated during the repair of chromosomal lesions. Recent work on Mus81 and Yen1/GEN1, two conserved structure-selective endonucleases, revealed unforeseen links between JM-processing and cell cycle progression. Cell cycle kinases and phosphatases control Mus81 and Yen1/GEN1 to restrain deleterious JM-processing during S-phase, while safeguarding chromosome segregation during mitosis. PMID:26970388

  4. ERK3 regulates TDP2-mediated DNA damage response and chemoresistance in lung cancer cells

    OpenAIRE

    Bian, Ka; Muppani, Naveen Reddy; Elkhadragy, Lobna; Wang, Wei; Zhang, Cheng; Chen, Tenghui; Jung, Sungyun; Seternes, Ole Morten; Long, Weiwen

    2015-01-01

    Posttranslational modifications (PTMs), such as phosphorylation and ubiquitination, play critical regulatory roles in the assembly of DNA damage response proteins on the DNA damage site and their activities in DNA damage repair. Tyrosyl DNA phosphodiesterase 2 (TDP2) repairs Topoisomerase 2 (Top2)-linked DNA damage, thereby protecting cancer cells against Top2 inhibitors-induced growth inhibition and cell death. The regulation of TDP2 activity by post-translational modifications in DNA repair...

  5. Cell cycle and DNA repair in UV-irradiated cells of mouse neuroblastoma

    International Nuclear Information System (INIS)

    A correlation has been shown between a reduced rate of movement of UV-irradiated neuroblastoma cells from G1 into S phase, an essential increase of cells in S phase while progressing through the cell cycle, and a defect in free DNA synthesis on a damaged template. These indices may reflect one and the same cell response to the UV light

  6. Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-06-01

    Full Text Available Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

  7. DNA damage in human endothelial cells after irradiation in anoxia

    International Nuclear Information System (INIS)

    Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from 60Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells. (orig.)

  8. Oxidative Stress Induces Mitochondrial DNA Damage and Cytotoxicity through Independent Mechanisms in Human Cancer Cells

    OpenAIRE

    Yue Han; Chen, Junjian Z.

    2013-01-01

    Intrinsic oxidative stress through increased production of reactive oxygen species (ROS) is associated with carcinogenic transformation, cell toxicity, and DNA damage. Mitochondrial DNA (mtDNA) is a natural surrogate to oxidative DNA damage. MtDNA damage results in the loss of its supercoiled structure and is readily detectable using a novel, supercoiling-sensitive real-time PCR method. Our studies have demonstrated that mtDNA damage, as measured by DNA strand breaks and copy number depletion...

  9. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  10. Immunopathological mechanisms of human T cell lymphotropic virus type 1 (HTLV-I) uveitis. Detection of HTLV-I-infected T cells in the eye and their constitutive cytokine production.

    OpenAIRE

    Sagawa, K; Mochizuki, M; Masuoka, K; Katagiri, K; Katayama, T.; T. Maeda; Tanimoto, A; Sugita, S.; Watanabe, T.; Itoh, K.

    1995-01-01

    The immunopathology of human T cell lymphotropic virus type 1 (HTLV-I) uveitis was addressed by using T cell clones (TCC) established from the intraocular fluid of patients with HTLV-I uveitis. Proviral DNA of HTLV-I was identified in 55 out of 94 (59%) or 13 out of 36 (36%) TCC from the ocular fluid or the peripheral blood of these patients, respectively. Most of HTLV-I-infected TCC had a CD3+ CD4+ CD8- phenotype. HTLV-I infection on TCC was confirmed by analysis of the viral mRNA, nucleotid...

  11. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott;

    2014-01-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial...... rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional...... DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly...

  12. Cell-free circulating tumor DNA in cancer.

    Science.gov (United States)

    Qin, Zhen; Ljubimov, Vladimir A; Zhou, Cuiqi; Tong, Yunguang; Liang, Jimin

    2016-01-01

    Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason underlying difficulties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive "liquid biopsy" from the blood has been attempted to characterize tumor heterogeneity. This review focuses on cell-free circulating tumor DNA (ctDNA) in the bloodstream as a versatile biomarker. ctDNA analysis is an evolving field with many new methods being developed and optimized to be able to successfully extract and analyze ctDNA, which has vast clinical applications. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. In addition, ctDNA has the potential to accurately monitor tumor burden and treatment response, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse. There are still many challenges that need to be overcome prior to this biomarker getting wide adoption in the clinical world, including optimization, standardization, and large multicenter trials. PMID:27056366

  13. ADP-ribosylation is involved in the integration of foreign DNA into the mammalian cell genome.

    OpenAIRE

    Farzaneh, F.; Panayotou, G N; Bowler, L. D.; Hardas, B D; Broom, T; Walther, C; Shall, S

    1988-01-01

    The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenat...

  14. Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

    OpenAIRE

    DeLange, A. M.; McFadden, G

    1986-01-01

    A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analy...

  15. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  16. DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

    International Nuclear Information System (INIS)

    Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

  17. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

    OpenAIRE

    Zuber, M; Tan, E M; Ryoji, M

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replicatio...

  18. Functionalization of DNA Nanostructures for Cell Signaling Applications

    Science.gov (United States)

    Pedersen, Ronnie O.

    Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by

  19. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

    Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

  20. DNA methylation arrays as surrogate measures of cell mixture distribution

    Directory of Open Access Journals (Sweden)

    Houseman Eugene

    2012-05-01

    Full Text Available Abstract Background There has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls. Results Here we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach. Conclusions Our method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

  1. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    Energy Technology Data Exchange (ETDEWEB)

    Lushaj, Entela B., E-mail: lushaj@surgery.wisc.edu [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States); Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States)

    2012-06-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  2. Cell Free DNA of Tumor Origin Induces a ‘Metastatic’ Expression Profile in HT-29 Cancer Cell Line

    OpenAIRE

    Fűri, István; Kalmár, Alexandra; Wichmann, Barnabás; Spisák, Sándor; Schöller, Andrea; Barták, Barbara; Tulassay, Zsolt; Molnár, Béla

    2015-01-01

    Background Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions. Aims To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts. Materials and Methods...

  3. Cell Adhesion Geometry Regulates Non-Random DNA Segregation and Asymmetric Cell Fates in Mouse Skeletal Muscle Stem Cells

    OpenAIRE

    Siham Yennek; Mithila Burute; Manuel Théry; Shahragim Tajbakhsh

    2014-01-01

    Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-rand...

  4. Cell Adhesion Geometry Regulates Non-Random DNA Segregation and Asymmetric Cell Fates in Mouse Skeletal Muscle Stem Cells

    OpenAIRE

    Yennek, Siham; Burute, Mithila; Théry, Manuel; Tajbakhsh, Shahragim

    2014-01-01

    International audience Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole posi...

  5. Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells.

    OpenAIRE

    Yennek, Siham; Burute, Mithila; Théry, Manuel; Tajbakhsh, Shahragim

    2014-01-01

    International audience Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole posi...

  6. Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells

    OpenAIRE

    Yennek, Siham; Burute, Mithila; Thery, Manuel

    2014-01-01

    Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-rand...

  7. Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

    Science.gov (United States)

    Medveczky, P; Kramp, W J; Sullivan, J L

    1984-11-01

    Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization with a cloned viral DNA fragment. The size of the viral DNA was estimated at ca. 158 kilobase pairs. Restriction endonuclease patterns suggested structural similarities to cottontail herpesvirus DNA. PMID:6092696

  8. Recombination events during integration of transfected DNA into normal human cells.

    OpenAIRE

    Murnane, J P; Yezzi, M J; Young, B. R.

    1990-01-01

    The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments contain...

  9. Bleomycin-induced DNA synthesis in a cell-free system using a permeable mouse sarcoma cell Extract.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1987-10-01

    Full Text Available To investigate factors involved in excision repair DNA synthesis, a soluble extract was prepared from permeable mouse sarcoma (SR-C3H/He cells by homogenization and ultracentrifugation. DNA synthesis measured by using native calf thymus DNA as the template-primer and the extract as the polymerase source showed low activity. The DNA synthesis was enhanced more than ten-fold by the addition of an appropriate concentration of bleomycin, a radiomimetic DNA-damaging drug. Using selective inhibitors of DNA polymerases, it was shown that the DNA polymerase involved in the bleomycin-induced DNA synthesis was DNA polymerase beta. In addition to DNA polymerase beta, an exonuclease which converts bleomycin-damaged DNA into suitable template-primers for repair DNA synthesis appeared to be present in the permeable cell extract.

  10. DNA Damage Response in Hematopoietic Stem Cell Ageing.

    Science.gov (United States)

    Li, Tangliang; Zhou, Zhong-Wei; Ju, Zhenyu; Wang, Zhao-Qi

    2016-06-01

    Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing. PMID:27221660

  11. DNA Damage Response in Hematopoietic Stem Cell Ageing

    Institute of Scientific and Technical Information of China (English)

    Tangliang Li; Zhong-Wei Zhou; Zhenyu Ju; Zhao-Qi Wang

    2016-01-01

    Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employ-ing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically reg-ulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing.

  12. Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells.

    Science.gov (United States)

    Ovádeková, Renáta; Jantová, Sona; Letasiová, Silvia; Stepánek, Ivan; Labuda, Ján

    2006-12-01

    Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP-MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker Co[(phen)3]3+ and the guanine moiety anodic oxidation and cyclic voltammetry with K3[Fe(CN)6] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 microg mL(-1). PMID:17053918

  13. NLP-1: a DNA intercalating hypoxic cell radiosensitizer and cytotoxin

    International Nuclear Information System (INIS)

    The 2-nitroimidazole linked phenanthridine, NLP-1 (5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide), was synthesized with the rationale of targeting the nitroimidazole to DNA via the phenanthridine ring. The drug is soluble in aqueous solution (greater than 25 mM) and stable at room temperature. It binds to DNA with a binding constant 1/30 that of ethidium bromide. At a concentration of 0.5 mM, NLP-1 is 8 times more toxic to hypoxic than aerobic cells at 37 degrees C. This concentration is 40 times less than the concentration of misonidazole, a non-intercalating 2-nitroimidazole, required for the same degree of hypoxic cell toxicity. The toxicity of NLP-1 is reduced at least 10-fold at 0 degrees C. Its ability to radiosensitize hypoxic cells is similar to misonidazole at 0 degrees C. Thus the putative targeting of the 2-nitroimidazole, NLP-1, to DNA, via its phenanthridine group, enhances its hypoxic toxicity, but not its radiosensitizing ability under the present test conditions. NLP-1 represents a lead compound for intercalating 2-nitroimidazoles with selective toxicity for hypoxic cells

  14. Study of stimulators of DNA synthesis in nerve tissue cells

    International Nuclear Information System (INIS)

    Changes in proliferative activity in different regions of the brain during ontogenesis are connected with changes in the composition and properties of regulators of cell proliferation. Extracts of regions of the brain in which active cell division takes place in a given stage of development (cortex of 15- to 17-day-old embryos or cerebellum of 8- to 10-day-old rats) can stimulate the incorporation of labeled precursors into the brain cell DNA of both newborn and adult rats. Salting out at increasing ammonium sulfate concentrations, gel filtration on Sephadex, and isoelectric focusing led to the isolation of three fractions of stimulators of DNA synthesis: in acid, neutral, and alkaline pH regions. A method is described for obtaining purified preparations and for determining some physicochemical properties of the acid activator, which is a low-molecular-weight peptide capable of noticeably stimulating the incorporation of labeled precursors into the DNA of nerve tissue cells when added to an in vitro system in a concentration of the order of 1 μg/ml

  15. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. PMID:24861204

  16. File list: Oth.ALL.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.DNA-RNA_hybrids.AllCell.bed ...

  17. File list: Oth.ALL.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.DNA-RNA_hybrids.AllCell.bed ...

  18. File list: Oth.ALL.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.DNA-RNA_hybrids.AllCell.bed ...

  19. File list: Oth.ALL.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.DNA-RNA_hybrids.AllCell.bed ...

  20. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Clement G. Yedjou

    2015-12-01

    Full Text Available In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO32] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60 cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO32 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05 increase of necrotic cell death in Pb(NO32-treated cells, indicative of membrane rupture by Pb(NO32 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05 in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO32 exposure significantly (p < 0.05 increased the proportion of caspase-3 positive cells (apoptotic cells compared to the control. The flow cytometry assessment also indicated Pb(NO32 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO32 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO32 exposure and its associated adverse

  1. Ultraviolet-induced cell death is independent of DNA replication in rat kangeroo cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyaji, E.N.; Menck, C.F.M. [Sao Paulo Univ., SP (Brazil). Inst. de Biociencias

    1995-05-01

    Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication. (author).

  2. Ultraviolet-induced cell death is independent of DNA replication in rat kangeroo cells

    International Nuclear Information System (INIS)

    Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication. (author)

  3. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    OpenAIRE

    Rolletschek Alexandra; Solozobova Valeriya; Blattner Christine

    2009-01-01

    Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells...

  4. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    OpenAIRE

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately c...

  5. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    OpenAIRE

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood m...

  6. Role of DNA deletion length in mutation and cell survival

    International Nuclear Information System (INIS)

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  7. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    OpenAIRE

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for ...

  8. Gene shuttling: moving of cloned DNA into and out of eukaryotic cells.

    OpenAIRE

    Lindenmaier, W; Hauser, H.; de Wilke, I G; Schütz, G

    1982-01-01

    Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant contain...

  9. DNA methylation changes in cells regrowing after fractioned ionizing radiation

    International Nuclear Information System (INIS)

    Background and purpose: Repeated exposure to ionizing radiation (IR) can result in adaptive reactions. While DNA methylation changes in adaption to repeated stress exposure are established for a variety of drugs, their role in fractioned ionizing radiation is largely unknown. Material and methods: MCF7 breast cancer cells were treated 5 times a week with IR in fractions of 2 Gy, resulting in total doses of 10 and 20 Gy. Cells were harvested 48 and 72 h after the last irradiation, as well as after a recovery period of at least 14 d. To identify genes differentially methylated in irradiated versus non-irradiated cells, we used methyl-CpG immunoprecipitation (MCIp) followed by global methylation profiling on CpG island microarrays. Results: MCIp profiling revealed methylation changes in several CpG islands 48 h after FIR with 10 and 20 Gy. Cells receiving a total dose of 10 Gy started regrowing after 14 d and exhibited similar radioresistance as mock-treated cells. Differential methylation of the CpG units associated with FOXC1 (p < 0.001) and TRAPPC9 (p < 0.001) could be confirmed by time-of-flight mass spectrometry (Sequenom). Conclusions: In summary, these data indicate that regrowth of MCF7 cells after 10 Gy FIR is associated with locus-specific alterations in DNA methylation.

  10. The studies of DNA metabolic dynamics in embryonic cell line and non-embryonic cell line of Onobrychis viciaefolia scop

    International Nuclear Information System (INIS)

    The hypocotyls of aseptic seedlings of Onobrychis viciaefolia Scop. were used as explants for inducing callus. The embryonic cell line (E-line) and non-embryonic cell line (NE line) were established. The DNA synthesis dynamics in both cell lines were studied by autoradiography. The results showed that: DNA synthesis in E-line was active and confined to embryonic cell or cell masses and then the cells divided quickly and formed somatic embryos at different stages. The changes of DNA metabolism took place in a certain pattern and the maximum rate of DNA synthesis occured during the formation of globular embryo. There was a clear relationship between the difference of DNA synthesis rate and the polarity of the embryo. In NE-line, the beginning of cell division and the forming of callus were also based on DNA replication but were much deoxythymidine. There was a significant difference in DNA synthesis dynamics between the two cell lines

  11. Genetic modelling of PIM proteins in cancer: proviral tagging, cooperation with oncogenes, tumor suppressor genes and carcinogens.

    Directory of Open Access Journals (Sweden)

    CarmenBlanco Aparicio

    2014-05-01

    Full Text Available The PIM proteins, which were initially discovered as proviral insertion sites in Moloney murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anticancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim and a third group of genes (including bmi1 and gfi1 as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all 3 isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis.

  12. A novel cell permeable DNA replication and repair marker.

    Science.gov (United States)

    Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

    2014-01-01

    Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25484186

  13. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  14. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-12-29

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

  15. DNA damage in Human Limbal Epithelial Cells expanded ex vivo.

    Directory of Open Access Journals (Sweden)

    Yolanda Lorenzo Corrales

    2015-04-01

    Full Text Available Limbal stem cell deficiency, secondary to insults and diseases, may be treated by transplantation of ex vivo engineered epithelial grafts. We here present preliminary data on levels of cellular DNA damage in grafts produced in two different types of culture medium. Cultures were initiated using corneo-limbal donor tissue after removal of the central area for transplant purposes. Explants (approx. 2x2 mm were positioned epithelial side down on tissue culture treated polyester membranes and expanded for four weeks in Dulbecco’s Modified Eagle Medium F12 Nutrient Mixture (Ham [DMEM/F12 (1:1] with either (1 H. medium; 10% human serum or (2 COM; 5% fetal bovine serum (FBS, Epidermal Growth Factor (EGF, insulin-transferrin-sodiumselenzine (ITS , cholera toxin-A, dimethyl sulfoxide (DMSO and hydrocortisone. Cells were dissociated using Trypsin-EDTA (0.05% for 30 min., the enzyme activity was inhibited by medium and serum. The cell suspension was transferred to tubes on ice and processed using the Comet Assay. Duplicate samples from each culture were analyzed in each assay by visual scoring. Using a fluorescence microscope, 100 comets (50 from each gel were classified into five categories, 0-4, representing increasing relative tail intensities. Summing the scores (0-4 of 100 comets therefore gives an overall score of between 0 and 400 arbitrary units. Preliminary data show some levels of DNA damage in cells dissociated from the grafts regardless of the type of culture medium used. Anyway more experiments with other donors have to be done to have some conclusions. Recent studies have shown that medium with human serum equally support production of grafts containing differentiated as well as undifferentiated cells suitable for clinical transplantation. Preliminary data from our experiments indicate that levels of molecular damage to the DNA do not increase in cells cultured in H. medium despite its lacks of complexity.

  16. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  17. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    International Nuclear Information System (INIS)

    Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

  18. Live cell microscopy of DNA damage response in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina; Gallina, Irene; Eckert-Boulet, Nadine Valerie; Lisby, Michael

    Fluorescence microscopy of the DNA damage response in living cells stands out from many other DNA repair assays by its ability to monitor the response to individual DNA lesions in single cells. This is particularly true in yeast, where the frequency of spontaneous DNA lesions is relatively low...... live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction of...

  19. Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR

    OpenAIRE

    White, Irene E.; Campbell, Thomas B.

    2000-01-01

    A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 105 copies of KSHV DNA (r2 > 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log10 copies for samples that contained from 10 to 105 copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10−5, and cell-free KSHV DNA in plasma was quantitated ...

  20. Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

    OpenAIRE

    Medveczky, P; Kramp, W J; Sullivan, J L

    1984-01-01

    Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization...

  1. Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process

    OpenAIRE

    Susanna Schubert; Sandra Heller; Birgit Löffler; Ingo Schäfer; Martina Seibel; Gaetano Villani; Peter Seibel

    2015-01-01

    Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By a...

  2. Replication independent formation of extrachromosomal circular DNA in mammalian cell-free system.

    Directory of Open Access Journals (Sweden)

    Zoya Cohen

    Full Text Available Extrachromosomal circular DNA (eccDNA is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence-independent enzymes as human protein extract can produce mouse-specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg(2+ and is enhanced by double strand DNA breaks.

  3. Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

    OpenAIRE

    Terazono Hideyuki; Anzai Yu; Soloviev Mikhail; Yasuda Kenji

    2010-01-01

    Abstract A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by ...

  4. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  5. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

    OpenAIRE

    Itoh, G; Tamura, J; M. Suzuki; Suzuki, Y.; Ikeda, H; Koike, M; Nomura, M; Jie, T; Ito, K

    1995-01-01

    Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end...

  6. Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells.

    Science.gov (United States)

    Yennek, Siham; Burute, Mithila; Théry, Manuel; Tajbakhsh, Shahragim

    2014-05-22

    Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates. PMID:24836002

  7. Cell Adhesion Geometry Regulates Non-Random DNA Segregation and Asymmetric Cell Fates in Mouse Skeletal Muscle Stem Cells

    Directory of Open Access Journals (Sweden)

    Siham Yennek

    2014-05-01

    Full Text Available Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates.

  8. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    Science.gov (United States)

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. PMID:26914280

  9. Induction of DNA damage by deguelin is mediated through reducing DNA repair genes in human non-small cell lung cancer NCI-H460 cells

    OpenAIRE

    JI, BIN-CHUAN; Chien-chih YU; YANG, SU-TSO; Hsia, Te-Chun; Yang, Jai-Sing; Lai, Kuang-Chi; Ko, Yang-Ching; Lin, Jen-Jyh; Lai, Tung-Yuan; Chung, Jing-Gung

    2012-01-01

    It has been shown that deguelin, one of the compounds of rotenoids from flavonoid family, induced cytotoxic effects through induction of cell cycle arrest and apoptosis in many types of human cancer cell lines, but deguelin-affected DNA damage and repair gene expression (mRNA) are not clarified yet. We investigated the effects of deguelin on DNA damage and associated gene expression in human lung cancer NCI-H460 cells in vitro. DNA damage was assayed by using the comet assay and DNA gel elect...

  10. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  11. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Svetlana Kostyuk

    2015-01-01

    Full Text Available Background. Cell free DNA (cfDNA circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci. As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR, PCNA (FACS and antiapoptotic genes (BCL2 (RT-PCR and FACS, BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR. Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs. Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR, in the level of fatty acid binding protein FABP4 (FACS analysis and in the level of fat (Oil Red O. Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  20. DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

    International Nuclear Information System (INIS)

    Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA)·(oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-αD-βD2, the two cell lines roughly have the same α value (∼0.40 Gy-1) whereas the β value was considerably higher in UM-SCC-14A (0.067 Gy-2 ± 0.007 Gy-2 [SEM]) as compared to UM-SCC-1 (0.013 Gy-2 ± 0.004 Gy-2 [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A (p < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity

  1. Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells.

    Directory of Open Access Journals (Sweden)

    Mori,Shigeru

    1989-04-01

    Full Text Available To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s.

  2. Effect of 5-fluorodeoxyuridine on DNA replication in ultraviolet-irradiated HeLa cells

    International Nuclear Information System (INIS)

    In HeLa cells precultivated for 6 hours with 5-fluorodeoxyuridine (FUdR) and for 18 hours in FUdR-free medium, DNA synthesis was much more resistant to UV irradiation than that of untreated cells. DNA synthesized in FUdR-pretreated and UV irradiated cells represents a semiconservative DNA replication and shows more rapid shift of the pulse-labelled chased DNA to high molecular weight. This DNA synthesis is not induced by synchronization of the cell cycle. It is assumed that either the changes of chromatine structure, or an enhanced level of some enzymes might be involved in the replication of the damaged template. (author)

  3. DNA Fragmentation in mammalian cells exposed to various light ions

    Science.gov (United States)

    Belli, M.; Cherubini, R.; Dalla Vecchia, M.; Dini, V.; Esposito, G.; Moschini, G.; Sapora, O.; Signoretti, C.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/μm protons, 123 keV/μm helium-4 ions and γ-rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respct to that induced by comparable doses of γ-rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for γ-rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage reparability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by γ-rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

  4. Characterization of DNA Methylation in Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    Constantin F. Pixberg

    2015-10-01

    Full Text Available Epigenetics contributes to molecular mechanisms leading to tumor cell transformation and systemic progression of cancer. However, the dynamics of epigenetic remodeling during metastasis remains unexplored. In this context, circulating tumor cells (CTCs might enable a direct insight into epigenetic mechanisms relevant for metastasis by providing direct access to systemic cancer. CTCs can be used as prognostic markers in cancer patients and are regarded as potential metastatic precursor cells. However, despite substantial technical progress, the detection and molecular characterization of CTCs remain challenging, in particular the analysis of DNA methylation. As recent studies have started to address the epigenetic state of CTCs, we discuss here the potential of such investigations to elucidate mechanisms of metastasis and to develop tumor biomarkers.

  5. DNA From Dead Cancer Cells Induces TLR9-Mediated Invasion and Inflammation In Living Cancer Cells

    Science.gov (United States)

    Tuomela, Johanna; Sandholm, Jouko; Kaakinen, Mika; Patel, Ankita; Kauppila, Joonas H.; Ilvesaro, Joanna; Chen, Dongquan; Harris, Kevin W.; Graves, David; Selander, Katri S.

    2014-01-01

    TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple negative, human MDA-MB-231 breast cancer cells stably expressing control or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy. PMID:24212717

  6. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    International Nuclear Information System (INIS)

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing

  7. Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content

    OpenAIRE

    sprotocols

    2014-01-01

    Author: Ingrid Schmid Corresponding author ([]()) ### INTRODUCTION Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and c...

  8. Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

    Institute of Scientific and Technical Information of China (English)

    DA-LIN HU; JIAN-PING YANG; DAO-KUI FANG; YAN SHA; XIAO-ZHI TU; ZHI-XIONG ZHUANG; HUAN-WEN TANG; HAI-RONG LIANG; DONG-SHENG TANG; YI-MING LIU; WEI-DONG JI; JIAN-HUI YUAN; YUN HE; ZHENG-YU ZHU

    2007-01-01

    Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-Cl were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 μmol/L to 120 μmol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. Results MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

  9. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    International Nuclear Information System (INIS)

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present

  10. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    OpenAIRE

    Abel Jacobus Bronkhorst; Janine Aucamp; Piet J. Pretorius

    2015-01-01

    Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identi...

  11. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  12. Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

    Directory of Open Access Journals (Sweden)

    Yuh Shiwa

    Full Text Available Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03 when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50 when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14 by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45 and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17. These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

  13. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    Science.gov (United States)

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  14. An antiretroviral drug-naïve human immunodeficiency virus-1 infected woman with a persistent non-reactive proviral deoxyribonucleic acid polymerase chain reaction: a case report

    OpenAIRE

    Kfutwah, Anfumbom KW; Ngono, Valerie; Ngoupo, Paul Alain; Njouom, Richard

    2013-01-01

    Introduction Replication of the human immunodeficiency virus involves an obligatory step of reverse transcription of the viral ribonucleic acid genome into a double-stranded deoxyribonucleic acid, and subsequent integration of the deoxyribonucleic acid into the human chromatin to form the proviral deoxyribonucleic acid. This proviral human immunodeficiency virus deoxyribonucleic acid is a critical marker for the diagnosis of acute infections, mother-to-child transmissions and for the confirma...

  15. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2009-06-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with {sup 137}Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H{sub 2}O{sub 2}-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H{sub 2}O{sub 2}-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

  16. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    Science.gov (United States)

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  17. DNA damaging and cell cycle effects of the topoisomerase I poison camptothecin in irradiated human cells

    International Nuclear Information System (INIS)

    This study addressed the potential radiosensitizing and DNA-damaging actions of the DNA topoisomerase I poison camptothecin (CPT) on SV40 transformed normal (MRC5CVI) and ataxia-telangiectasia (AT5BIVA) fibroblast cell lines. In both cell lines CPT induced a dose-dependent delay of cells in S phase, followed by a dose-dependent trapping in G2/M phase. Acute X-irradiation produced patterns of G2/M arrest and S-phase delay similar to those observed for CPT in the MRC5CVI cell line, but no S phase delay was observed in the AT5BIVA cell line consistent with the ataxia-telangiectasia phenotype of this cell line. X-irradiation of CPT-treated cells resulted in additive prolongation of S phase delay in MRC5CVI cultures and additive effects for cell killing in both cell lines. The potential for topoisomerase I-DNA cross-linking by CPT was not altered by 24 h pretreatment with CPT, or by acute X-irradiation. Hypersensitivity of AT5BIVA to CPT was not attributable to elevated levels of complex trapping. (author)

  18. Complete in vitro DNA replication of SV40 chromatin in digitonin-treated permeable cells.

    OpenAIRE

    Oda,Takuzo; Watanabe,Sekiko; Hanakawa,Shiro; Nakamura, Takashi

    1980-01-01

    A permeable cell system has been developed by treatment with digitonin for studying in vitro DNA replication of chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in digitonin-treated permeable cells was analyzed by electrophoresis in agarose-gel. Autoradiography of the agarose-gel revealed that [32P]dCTP was incorporated in SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated the complete replication of SV40 DNA and ...

  19. Associations between cigarette smoking and mitochondrial DNA abnormalities in buccal cells

    OpenAIRE

    Tan, Duanjun; Goerlitz, David S.; Dumitrescu, Ramona G.; Han, Dingfen; Seillier-Moiseiwitsch, Françoise; Spernak, Stephanie M.; Orden, Roy Anthony; Chen, Jinguo; Goldman, Radoslav; Shields, Peter G.

    2008-01-01

    DNA alterations in mitochondria are believed to play a role in carcinogenesis and are found in smoking-related cancers. We sought to replicate earlier findings for the association of smoking with increased mitochondrial DNA (mtDNA) content in buccal cells and further hypothesized that there would be an increased number of somatic mtDNA mutations in smokers. Buccal cells and blood lymphocytes were studied from 42 healthy smokers and 30 non-smokers. Temporal temperature gradient electrophoresis...

  20. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  1. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment. PMID:20596959

  2. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    Science.gov (United States)

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. Conclusion: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results. PMID:26140187

  3. Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

    International Nuclear Information System (INIS)

    Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal

  4. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    Directory of Open Access Journals (Sweden)

    Eun Ah Song

    2016-08-01

    Full Text Available The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA, shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

  5. DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this ''contact effect'' to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation

  6. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  7. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  8. Detection of DNA damage in cells exposed to ionizing radiation by use of antisingle-stranded-DNA monoclonal antibody

    International Nuclear Information System (INIS)

    An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody, D1B, was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the partial umwinding of cellular DNA under alkaline conditions, a time-dependent process. Single-strand and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding under controlled conditions is a measure, therefore, of the amount of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gu of ionizing radiation and needs only small amounts of cells. The usefulness of the technique was demonstrated in a study on the induction of damage and its repair in unlabelled cultured Chinese hamster cells and in DNA-containing cells of human blood, both after exposure to 60Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related degree of unwinding was observed and repair could be observed up to 60 min after irradiation. (author). 19 refs.; 3 figs.; 1 tab

  9. Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents

  10. TUMOR-RELATED METHYLATED CELL-FREE DNA AND CIRCULATING TUMOR CELLS IN MELANOMA

    Directory of Open Access Journals (Sweden)

    Francesca eSalvianti

    2016-01-01

    Full Text Available Solid tumor release into the circulation cell-free DNA (cfDNA and circulating tumor cells (CTCs which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma.The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs.RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC.The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic than in healthy subjects (Pearson chi-squared test, p<0.001. The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive

  11. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    Science.gov (United States)

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; De Giorgi, Vincenzo; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and

  12. The novel immunosuppressive protein kinase C inhibitor sotrastaurin has no pro-viral effects on the replication cycle of hepatitis B or C virus.

    Directory of Open Access Journals (Sweden)

    Thomas von Hahn

    Full Text Available The pan-protein kinase C (PKC inhibitor sotrastaurin (AEB071 is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV and hepatitis C virus (HCV, major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients.

  13. Comparison of DNA double—strand breaks induced by 16O8+ in deproteinized DNA and intact cells

    Institute of Scientific and Technical Information of China (English)

    ZhouGuang-Ming; GaoQing-Xiang; 等

    1998-01-01

    The yield of DNA double-strand breaks(DSBs) is sure to be influenced by the environment around DNA molecule.Inverse pulsed-field gel electrophoresis(PIGE)has been applied to compared the sensitivity of B16 cells and their DNA in DSBs induced by 75MeV/u 16O8+ beam.Results show that the percentages of DNA released from the plug(PR)in both kinds of the samples increase with the dose and approach a similar quasi-threshold of about 81%.A simple new equation was presented to calculated the break level of DNA molecules.Within a certain dose,the relationship between the break level and the dose is linear.THe yield of DSBs in deproteinized DNA was 1.11DSBs/100Mbp/Gy,while that in intact cells was 0.60DSBs/100Mbp/Gy.it is testified that deproteinized DNA is more sensitive to oxygen ions irradiation than intact cells.

  14. The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

    Science.gov (United States)

    Bonjean, K; De Pauw-Gillet, M C; Defresne, M P; Colson, P; Houssier, C; Dassonneville, L; Bailly, C; Greimers, R; Wright, C; Quetin-Leclercq, J; Tits, M; Angenot, L

    1998-04-14

    Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor

  15. Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

    International Nuclear Information System (INIS)

    The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents

  16. Quantification of circulating cell-free DNA in the plasma of cancer patients during radiation therapy

    International Nuclear Information System (INIS)

    Cell-free plasma DNA is elevated in cancer patients and decreases in response to effective treatments. Consequently, these nucleic acids have potential as new tumor markers. In our current study, we investigated whether the plasma DNA concentrations in patients with cancer are altered during the course of radiation therapy. To first determine the origin of cell-free plasma DNA, plasma samples from mice bearing transplanted human tumors were analyzed for human-specific and mouse-specific cell-free DNA. Human-specific DNA was detectable only in plasma from tumor-bearing mice. However, mouse-specific plasma DNA was significantly higher in tumor-bearing mice than in normal mice, suggesting that cell-free plasma DNA originated from both tumor and normal cells. We measured the total cell-free plasma DNA levels by quantitative polymerase chain reaction in 15 cancer patients undergoing radiation therapy and compared these values with healthy control subjects. The cancer patients showed higher pretreatment plasma DNA concentrations than the healthy controls. Eleven of these patients showed a transient increase of up to eightfold in their cell-free plasma DNA concentrations during the first or second week of radiation therapy, followed by decreasing concentrations toward the end of treatment. In two other cancer patients, the cell-free plasma DNA concentrations only decreased over the course of the treatment. The total cell-free plasma DNA levels in cancer patients thus show dynamic changes associated with the progression of radiation therapy. Additional prospective studies will be required to elucidate the potential clinical utility and biological implications of dynamic changes in cell-free plasma DNA during radiation therapy. (author)

  17. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells

    Directory of Open Access Journals (Sweden)

    Gersende Caron

    2015-11-01

    Full Text Available Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxymethylation, and cell fate determination.

  18. Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

    Science.gov (United States)

    Devaney, Stephanie A.; Palomaki, Glenn E.; Scott, Joan A.; Bianchi, Diana W.

    2015-01-01

    Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997–April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%–96.1%) and specificity, 98.6% (95% CI, 98.1%–99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989–0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979–0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993–0.998) for real-time quantitative PCR (RTQ-PCR) (P=.02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965–0.998) (20 weeks) (P=.02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96

  19. HIV-1 Tat depresses DNA-PKCS expression and DNA repair, and sensitizes cells to ionizing radiation

    International Nuclear Information System (INIS)

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and γ-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and γH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy γ-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G2/M arrest as compared with parental cells. In addition, the G2/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS

  20. ShaPINg cell fate upon DNA damage:role of Pin1 isomerase in DNA damage-induced cell death and repair

    Directory of Open Access Journals (Sweden)

    Thomas G Hofmann

    2014-06-01

    Full Text Available The peptidyl-prolyl cis/trans isomerase Pin1 acts as a molecular timer in proline-directed Ser/Thr kinase signaling and shapes cellular responses based on recognition of phosphorylation marks and implementing conformational changes in its substrates. Accordingly, Pin1 has been linked to numerous phosphorylation-controlled signaling pathways and cellular processes such as cell cycle progression, proliferation and differentiation. In addition, Pin1 plays a pivotal role in DNA damage-triggered cell fate decisions. Whereas moderate DNA damage is balanced by DNA repair, cells confronted with massive genotoxic stress are eliminated by the induction of programmed cell death or cellular senescence. In this review we summarize and discuss the current knowledge on how Pin1 specifies cell fate through regulating key players of the apoptotic and the repair branch of the DNA damage response.

  1. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    OpenAIRE

    Razmik Mirzayans; Bonnie Andrais; Piyush Kumar; David Murray

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and t...

  2. Self DNA from lymphocytes that have undergone activation-induced cell death enhances murine B cell proliferation and antibody production.

    Directory of Open Access Journals (Sweden)

    Qing Lu

    Full Text Available Systemic lupus erythematosus (SLE is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells in vitro. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation in vitro and in vivo. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138+ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1 and the X-box binding protein 1 (XBP1. Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.

  3. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    OpenAIRE

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2011-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excisio...

  4. DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM

    Science.gov (United States)

    DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanismAnuradha Mudipalli.Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

  5. Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

    DEFF Research Database (Denmark)

    Bechmann, Troels; Andersen, Rikke Fredslund; Pallisgaard, Niels;

    2013-01-01

    Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in...... cfDNA during neoadjuvant chemotherapy....

  6. Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals

    DEFF Research Database (Denmark)

    Møller, Peter; Jensen, Ditte Marie; Christophersen, Daniel Vest;

    2015-01-01

    -reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations...

  7. [An analysis of the DNA synthesized in adenovirus-infected cells under exposure to nucleoside analogs].

    Science.gov (United States)

    Nosach, L N; Butenko, S I; Timofeeva, M Ia; Diachenko, N S; Tikhomirova, T P

    1989-01-01

    The method of dot DNA-DNA hybridization was used to reveal the inhibition of the synthesis of the adenoviral DNA by 6-azacytidine, cyclocytidine and ribamidyl in the adenovirus-infected cells Hep-2, a degree of which depended on the preparation concentration. PMID:2482929

  8. The Clinical Utilization of Circulating Cell Free DNA (CCFDNA in Blood of Cancer Patients

    Directory of Open Access Journals (Sweden)

    Yanyuan Wu

    2013-09-01

    Full Text Available Qualitative and quantitative testing of circulating cell free DNA (CCFDNA can be applied for the management of malignant and benign neoplasms. Detecting circulating DNA in cancer patients may help develop a DNA profile for early stage diagnosis in malignancies. The technical issues of obtaining, using, and analyzing CCFDNA from blood will be discussed.

  9. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dizdaroglu, Miral

    1999-05-12

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  10. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    International Nuclear Information System (INIS)

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee ampersand Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of ''naked DNA'' for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  11. Radiation-induced depression of DNA synthesis in cultured mammalian cells

    International Nuclear Information System (INIS)

    A 313-nm light source was constructed in order to study the mechanisms by which ultraviolet and ionizing radiations inhibit DNA synthesis. It was found that in CHO, MDBK and HeLa cells, grown for one generation in the DNA sensitizer bromodeoxyuridine (BrdUrd), 313-nm light inhibited DNA synthesis with a pattern similar to that of the effect of x-rays on normal cells. A biphasic dose response curve for inhibition of total synthesis was observed, with a sensitive component representing depression of initiation of new replicons and a resistant component representing interference with elongation of replicons already growing at the time of irradiation. Since the BrdUrd plus 313-nm light treatment produces DNA lesions similar to those produced by x-rays (base damage, strand breaks, crosslinks) these results suggest that the effect of x-rays on DNA synthesis is mediated by DNA damage. In experiments with synchronized cells, it was found that in cells in which about half the chromosomes had incorporated BrdUrd, 313-nm light inhibited replication of the BrdUrd-containing DNA, but had no effect on the replication of the unsubstituted DNA in the same cell. Thus the information that DNA is damaged appears to be propagated along the DNA molecule from the sites of damage to the replication initiation sites as some kind of conformational change, possibly a relaxation of superhelical tension. Target theory calculations suggest that a single DNA lesion prevents the initiation of several adjacent replicons

  12. Identification of DNA Methylation-Independent Epigenetic Events Underlying Clear Cell Renal Cell Carcinoma.

    Science.gov (United States)

    Becket, Elinne; Chopra, Sameer; Duymich, Christopher E; Lin, Justin J; You, Jueng Soo; Pandiyan, Kurinji; Nichols, Peter W; Siegmund, Kimberly D; Charlet, Jessica; Weisenberger, Daniel J; Jones, Peter A; Liang, Gangning

    2016-04-01

    Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR. PMID:26759245

  13. Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

    Institute of Scientific and Technical Information of China (English)

    Jianwei ZHEN; Qincl LIU; Jianhong GU; Yan YUAN; Xuezhong LIU; Handong WANG; Zongping LIU; Jianchun BIAN

    2012-01-01

    Abstract [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD~o) of ZEN with tetra- zolium-based colorimetric assay (MTT assay). Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group (P〈0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60_+4.78, 57.75_+6.25, 78.97_+5.83, 100.50~6.94 and 146.83_+12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_+2.25%, 22.24_+2.43%, 31.21_+ 6.27%, 37.45_+4.33% and 60.68_+9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences (P〈0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

  14. Role of DNA methylation in cell cycle arrest induced by Cr (VI in two cell lines.

    Directory of Open Access Journals (Sweden)

    Jianlin Lou

    Full Text Available Hexavalent chromium [Cr(IV], a well-known industrial waste product and an environmental pollutant, is recognized as a human carcinogen. But its mechanisms of carcinogenicity remain unclear, and recent studies suggest that DNA methylation may play an important role in the carcinogenesis of Cr(IV. The aim of our study was to investigate the effects of Cr(IV on cell cycle progress, global DNA methylation, and DNA methylation of p16 gene. A human B lymphoblastoid cell line and a human lung cell line A549 were exposed to 5-15 µM potassium dichromate or 1.25-5 µg/cm² lead chromate for 2-24 hours. Cell cycle was arrested at G₁ phase by both compounds in 24 hours exposure group, but global hypomethylation occurred earlier than cell cycle arrest, and the hypomethylation status maintained for more than 20 hours. The mRNA expression of p16 was significantly up-regulated by Cr(IV, especially by potassium dichromate, and the mRNA expression of cyclin-dependent kinases (CDK4 and CDK6 was significantly down-regulated. But protein expression analysis showed very little change of p16 gene. Both qualitative and quantitative results showed that DNA methylation status of p16 remained unchanged. Collectively, our data suggested that global hypomethylation was possibly responsible for Cr(IV-induced G₁ phase arrest, but DNA methylation might not be related to up-regulation of p16 gene by Cr(IV.

  15. FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2. (authors)

  16. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

    OpenAIRE

    Weingeist, David M.; Ge, Jing; Wood, David K.; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A.; Yaffe, Michael B.; Floyd, Scott; Engelward, Bevin P.

    2013-01-01

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluatio...

  17. The latest progress in single cell gel electrophoresis (SCGE) based on DNA damage detection

    International Nuclear Information System (INIS)

    DNA damage detection can detect DNA damage caused by the pesticide and irradiation. With the increasing demands of DNA damage detection, the development of a rapid, high throughput and straight forward DNA damage detecting technique has critical biological significance for Single Cell Gel Electrophoresis (SCGE) is a straight and accurate way to detect the DNA damage. In recent years, the throughput and accuracy of the detection SCGE method have been improved significantly by applying new materials and new technologies. This paper reviewed the most recently reported SCGE based DNA damage detection technique-microwell array method and conventional SCGE method, and the prospect were also discussed. (authors)

  18. Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases.

    Science.gov (United States)

    Clark-Ganheart, Cecily A; Iqbal, Sara N; Brown, Donna L; Black, Susan; Fries, Melissa H

    2014-05-01

    Circulating cell free fetal DNA (cffDNA) is an effective screening modality for fetal aneuploidy. We report two cases of false positive results. The first case involves a female, with self-reported Down syndrome. CffDNA returned positive for trisomy 18 leading to a maternal diagnosis of mosaicism chromosome 18 with normal fetal karyotype. The second case involves a patient with an anomalous fetal ultrasound and cffDNA positive for trisomy 13. Amniocentesis demonstrated a chromosome 8p duplication/deletion. False positive cffDNA may arise in clinical scenarios where diagnostic testing is clearly indicated. Practitioners should recognize the limitations of cffDNA. PMID:25298847

  19. Construction of a Sequencing Library from Circulating Cell-Free DNA.

    Science.gov (United States)

    Fang, Nan; Löffert, Dirk; Akinci-Tolun, Rumeysa; Heitz, Katja; Wolf, Alexander

    2016-01-01

    Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA. © 2016 by John Wiley & Sons, Inc. PMID:27038390

  20. Detection of apoptotic cells by selective precipitation of [3H]thymidine-labelled DNA.

    OpenAIRE

    Patki, A H; Lederman, M M

    1996-01-01

    Apoptosis is characterized by systematic fragmentation of high-molecular-weight DNA into oligonucleosome fragments. A sensitive method for detection of apoptotic cells involving [3H]thymidine-labelled DNA is presented. Cells from mid-log-phase cultures were labelled with [3H]thymidine for 15 to 18 h and then exposed to gamma irradiation to induce apoptosis. A modified Hirt method was used to separate low-molecular-weight DNA from high-molecular-weight DNA. The percentage of fragmented DNA and...

  1. Configuration of nucleolarDNA in situ in nucleolus ofAllium cepa cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The location and configuration of nucleolar DNA have not beendetermined for a long time. In this paper, we have observed the nucleolar ultrastructure and the character of nucleolar DNA in Allium cepa cells by conventional electron microscopy and the cytochemical NAMA-Ur DNA specific staining method. Furthermore, we have properly improved the NAMA-Ur method so as to analyze the location and configuration of nucleolar DNA in situ. Our results indicated that the nucleolar DNA in Allium cepa cells is mainly located at the border between fibrillar centers and dense fibrillar component, especially distributed in the configuration of encircling the fibrillar centers.

  2. Regulation of Anti-DNA B Cells in Recombination-activating Gene–deficient Mice

    OpenAIRE

    Xu, Hui; Li, Hui; Suri-Payer, Elisabeth; Hardy, Richard R.; Weigert, Martin

    1998-01-01

    Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B ce...

  3. Anti–DNA B Cells in MRL/lpr Mice Show Altered Differentiation and Editing Pattern

    OpenAIRE

    Li, Yijin; Li, Hui; Ni, Dongyao; Weigert, Martin

    2002-01-01

    We have studied the regulation of anti–DNA B cells in transgenic mice with a heavy chain transgene (3H9H/56R). This transgene codes for a heavy chain that forms anti–double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vκ repertoire, but certain L chains, referred to as Vκ editors, do not sustain dsDNA binding in combination with 3H9H/56R. In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is a...

  4. Methylation of cell-free circulating DNA in the diagnosis of cancer

    OpenAIRE

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular ca...

  5. PCR-based detection of a rare linear DNA in cell culture

    Directory of Open Access Journals (Sweden)

    Saveliev Sergei V.

    2002-01-01

    Full Text Available The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

  6. Ceruloplasmin reduces DNA double strand breaks and improves cell survival in lymphoblastoid cells

    International Nuclear Information System (INIS)

    Full text: Ionizing radiation through oxidative free radical production causes dose-dependent oxidative damage to biological macromolecules. To reduce the oxidative stress from ionizing radiation, use of antioxidants has been suggested as a prophylactic and early remedy of pathogenic therapy. Ceruloplasmin (Cp), a plasma protein produced by the liver, belongs to a class of multi copper ferroxidases known for their role in iron metabolism in vertebrates, including humans. Functions of Cp include copper transport, ferroxidase and aminooxidase activities. Serum Cp concentration fluctuates during inflammation, infection, trauma and irradiation. The role of Cp as an antioxidant after irradiation is not fully understood. Our aim was to investigate the radioprotective efficacy of Cp. We studied the effect of ceruloplasmin on the in-vitro radiosensitivity of lymphoblastoid cells lines after gamma-ray irradiation. We used radiosensitive cell lines LB0003, LB0004 and LB 0005, established from individuals who developed late radiation necrosis following curative radiotherapy and non-sensitive cell lines Masci and LB0001 as controls. The cell lines were irradiated with doses from 0 - 60 Gy. Genomic DNA was extracted at 0 - 24h after irradiation and subjected to PFGE to analyse the initial quantity of DNA DSBs and the quantity of unrepaired DSBs to evaluate the kinetics of DSB rejoining. Human Cp (0.05 or 0.5mg/ml) was added to cell cultures 30 min before or 5 min after irradiation. In the presence of Cp cell survival was increased and the level of DBSs reduced demonstrating its radioprotective effect and the potential mechanism of its protection against radiation effects. Its radioprotective efficacy on dose and time of administration of Cp. The radiosensitive cell lines differ from controls by kinetics of DSBs repair. Importantly, in presence of ceruloplasmin the level of DNA DSB was reduced and the kinetics of DNA DSB repair became comparable to that in controls

  7. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    Directory of Open Access Journals (Sweden)

    Abel Jacobus Bronkhorst

    2016-03-01

    Full Text Available Evaluating the kinetics of cell-free DNA (cfDNA in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA (“Cell-free DNA: Preanalytical variables” [1]. The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers.

  8. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Aucamp, Janine; Pretorius, Piet J.

    2015-01-01

    Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA (“Cell-free DNA: Preanalytical variables” [1]). The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers. PMID:26862578

  9. Improved recovery of bisulphite-treated cell-free DNA in plasma

    DEFF Research Database (Denmark)

    Pedersen, Inge Søkilde; Krarup, H.B.; Thorlacius-Ussing, O.;

    Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amount of cell-free DNA in plasma, sensitivity of the detection methods are of utmost importance. The vast majority of currently available methods for analysing DNA...... of PCR amplifying methylated and umethylated MEST. This procedure allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma....... methylation are based on bisulphite-mediated deamination of cytosine. However, the recovery of bisulphite-converted DNA is very poor. Here we introduce an alternative method for the crucial steps of bisulphite removal and desulfonation, improving recovery, especially for specimens with low levels of DNA. The...

  10. Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia

    Directory of Open Access Journals (Sweden)

    Mézes Miklós

    2009-01-01

    Full Text Available Abstract Background The aim of our study was to examine whether increased circulating total cell-free DNA levels are related to the clinical characteristics and standard laboratory parameters of preeclamptic patients, to markers of inflammation, endothelial activation or injury, oxidative stress and to cell-free fetal DNA levels. Methods Circulating total cell-free DNA was measured by real-time quantitative PCR in plasma samples obtained from 67 preeclamptic and 70 normotensive pregnant women. Standard laboratory parameters, C-reactive protein, plasma von Willebrand factor antigen, plasma fibronectin, plasma malondialdehyde and cell-free fetal DNA levels were also determined. Results and Conclusion Circulating total cell-free and fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (median: 11.395 vs. 32.460 and 0.001 vs. 0.086 pg/μl; P < .001. The quantity of plasma total cell-free DNA did not correlate with most of the laboratory parameters, except for serum aspartate aminotransferase and alanine aminotransferase activities (correlation coefficient: 0.31; P = 0.012 and 0.46; P < .001. There was no correlation with clinical characteristics, including body mass index. The releases of both free fetal and total cell-free deoxyribonucleic acid were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible - at least partly - for increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities.

  11. DNA synthesis and cell division in the adult primate brain

    International Nuclear Information System (INIS)

    It is generally accepted that the adult human brain is incapable of producing new neuron. Even cursory examination of neurologic, neuropathologic, or neurobiological textbooks published during the past 50 years will testify that this belief is deeply entrenched. In his classification of cell populations on the basis of their proliferative behavior, Leblond regarded neurons of the central nervous system as belonging to a category of static, nonrenewing epithelial tissue incapable of expanding or replenishing itself. This belief, however needs to re reexamined for two major reasons: First, as reviewed below, a number of reports have provided evidence of neurogenesis in adult brain of several vertebrate species. Second, the capacity for neurogenesis in the adult primate central nervous system has never been examined by modern methods. In this article the author described recent results from an extensive autoradiographic analysis performed on twelve rhesus monkeys injected with the specific DNA precursor [3H] thymidine at ages ranging from 6 postnatal months to 17 years

  12. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    OpenAIRE

    Yang Cao; Nicole L. Hoppman; Sarah E Kerr; Sattler, Christopher A.; Borowski, Kristi S.; Wick, Myra J.; Edward Highsmith, W.; Umut Aypar

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and speci...

  13. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia.

    Science.gov (United States)

    Bouquet, Fanny; Ousset, Marielle; Biard, Denis; Fallone, Frédérique; Dauvillier, Stéphanie; Frit, Philippe; Salles, Bernard; Muller, Catherine

    2011-06-01

    DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways. PMID:21576354

  14. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    International Nuclear Information System (INIS)

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [3H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

  15. In vivo measurement of DNA synthesis rates of colon epithelial cells in carcinogenesis

    International Nuclear Information System (INIS)

    We describe here a highly sensitive technique for measuring DNA synthesis rates of colon epithelial cells in vivo. Male SD rats were given 2H2O (heavy water). Colon epithelial cells were isolated, DNA was extracted, hydrolyzed to deoxyribonucleosides, and the deuterium enrichment of the deoxyribose moiety was determined by gas chromatographic/mass spectrometry. Turnover time of colon crypts and the time for migration of cells from basal to top fraction of the crypts were measured. These data were consistent with cell cycle analysis and bromodeoxyuridine labeling. By giving different concentrations of a promoter, dose-dependent increases in DNA synthesis rates were detected, demonstrating the sensitivity of the method. Administration of a carcinogen increased DNA synthesis rates cell proliferation in all fractions of the crypt. In conclusion, DNA synthesis rates of colon epithelial cells can be measured directly in vivo using stable-isotope labeling. Potential applications in humans include use as a biomarker for cancer chemoprevention studies

  16. Hydroxyurea does not prevent synchronized G1 Chinese hamster cells from entering the DNA synthetic period

    International Nuclear Information System (INIS)

    Using very high concentrations of radioactively labeled thymidine, we show that synchronized G1 cells treated with hydroxyurea entered the DNA synthetic period at a time and rate indistinguishable from that of untreated cells, although the rate of DNA synthesis was greatly reduced in the drug-treated cultures. The DNA synthesized in the presence of hydroxyurea was less than or equal to 1 x 107 daltons, all of which could be chased into bulk DNA of approximately 3.5 x 108 daltons within 3 hr after removal of hydroxyurea. Hydroxyurea synchronized cells are apparently not blocked at the G1/S boundary but in the S phase itself

  17. Modifying Risk of Aneuploidy with a Positive Cell-Free Fetal DNA Result.

    Science.gov (United States)

    Long, A Ashleigh; Abuhamad, Alfred Z; Warsof, Steven L

    2016-06-01

    Noninvasive genomic assessments of the fetus while in utero have been made possible by the analysis of cell-free fetal DNA fragments from the serum of pregnant women, as part of a noninvasive prenatal testing screening strategy. Between 7% and 10% of total cell-free DNA in the maternal blood comes from placental trophoblasts, allowing for identification of the DNA associated with the fetal component of the placenta. Using simple venipuncture in the outpatient setting, this cell-free, extracellular fetal DNA can be isolated in the maternal serum from a single blood draw as early as the seventh week of gestation. PMID:27235910

  18. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    OpenAIRE

    Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

    2015-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)...

  19. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    OpenAIRE

    Mahmoud Aljurf; Hala Abalkhail; Amal Alseraihy; Said Y. Mohamed; Mouhab Ayas; Fahad Alsharif; Hazza Alzahrani; Abdullah Al-Jefri; Ghuzayel Aldawsari; Ali Al-Ahmari; Belgaumi, Asim F.; Claudia Ulrike Walter; Hassan El-Solh; Walid Rasheed; Maher Albitar

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leuke...

  20. X-radiation induced double-strand DNA breaks in rat bone marrow cells

    International Nuclear Information System (INIS)

    The method of sedimentation in a neutral sucrose gradient was used to study y doublestranded dna in a total population of rat bone marrow cells. As a resul of cell lysis in neutral conditions the fragments of double-stranded dna were fo ormed having the molecular mass of (3+-0.3)x109D. A study was made of the dynamics of accumulation of dna double-strand breaks after irradiation of a cell l suspension. It was shown that the yield of double-strand breaks and ratio between single- and double-strand breaks in bone marrow cells were similar to th hose of cultured L5178Y cells

  1. DNA-electrophoresis of single cells - a method to screen for irradiated foodstuffs

    International Nuclear Information System (INIS)

    Microelectrophoresis of single cells can be used to detect γ-irradiation over a wide dose range and for a variety of products. It is a simple and rapid test for DNA damages and can be used for screening. The method was tested on cell suspensions of bone marrow and muscle cells from frozen chicken legs, chicken heart, turkey liver, beef and pork irradiated with doses up to 3 kGy. Cell suspensions were prepared by incubation of tissues in EDTA-SDS-buffer at pH 8. Single cell electrophoresis was performed in 0.75% agarose gel. DNA was visualised by silver staining. In unirradiated samples no or only a small amount of DNA penetrated the cell membranes. Cells of irradiated samples appeared like a ''comet'' due to to migration of DNA-fragments out of cell. (orig.)

  2. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan;

    2008-01-01

    Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem...... cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...... fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...

  3. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains

    OpenAIRE

    Jensen, Taylor J.; Kim, Sung K; Zhu, Zhanyang; Chin, Christine; Gebhard, Claudia; Lu, Tim; Deciu, Cosmin; Van den Boom, Dirk; Ehrich, Mathias

    2015-01-01

    Background Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. Results We perform whole genome bisulfite sequencing on a set of unmatched sampl...

  4. Relative ultraviolet radiation sensitivity of certain functions of polyoma virus. Stimulation of cell DNA synthesis

    International Nuclear Information System (INIS)

    Peritoneal Mouse macrophages were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivation of infectivity and of induction of DNA synthesis. By statistical analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis

  5. Processing of DNA for nonhomologous end-joining by cell-free extract

    OpenAIRE

    Budman, Joe; Chu, Gilbert

    2005-01-01

    In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of ...

  6. Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.

    Science.gov (United States)

    Rosado, Ivan V; Langevin, Frédéric; Crossan, Gerry P; Takata, Minoru; Patel, Ketan J

    2011-12-01

    Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival. PMID:22081012

  7. Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy

    OpenAIRE

    Olfat M. Hendy; Tawfik Abdel Motalib; Mona A. El Shafie; Fatma A. Khalaf; Sobhy E. Kotb; Aziza Khalil; Salwa R. Ali

    2016-01-01

    Background: Systemic lupus erythematosus (SLE) is the most heterogeneous chronic autoimmune disease; it is characterized by the presence of auto reactive B and T cells, responsible for the aberrant production of a broad and heterogeneous group of autoantibodies. Recent studies using various detection methods have demonstrated the elevations of circulating DNA in SLE patients. Aim of the study: The current study aimed to measure cell-free DNA (cf-DNA) in SLE patients as a potential tool to ...

  8. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  9. Reactivation of UV-irradiated plasmid transforming DNA by cells of yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Bekker, M.L.; Kozhina, T.N.; Smolina, V.S. (AN SSSR, Leningrad. Inst. Yadernoj Fiziki)

    1983-01-01

    Data revealing that cells of yeast Sccharomyces cerevisiae can reactivate transforming plasmid DNA after UV-radiation are given, this phenomenon at least partially depends on the system of exision reparation of master cells. Dependence of yeast survival rate and yield of yeast transformants on the UV-radiation dose of transforming DNA plasmid is disclosed.

  10. Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

    OpenAIRE

    Nocker, Andreas; Camper, Anne K.

    2006-01-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A prom...

  11. Nuclear Reorganization of Mammalian DNA Synthesis Prior to Cell Cycle Exit

    OpenAIRE

    Barbie, David A; Kudlow, Brian A.; Frock, Richard; Zhao, Jiyong; Johnson, Brett R.; Dyson, Nicholas; Harlow, Ed; Kennedy, Brian K.

    2004-01-01

    In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA sy...

  12. HERV-K and LINE-1 DNA methylation and reexpression in urothelial carcinoma

    Directory of Open Access Journals (Sweden)

    WolfgangGoering

    2013-09-01

    Full Text Available Changes in DNA methylation frequently accompany cancer development. One prominent change is an apparently genome-wide decrease in methylcytosine that is often ascribed to DNA hypomethylation at retroelements comprising nearly half the genome. DNA hypomethylation may allow reactivation of retroelements, enabling retrotransposition and causing gene expression disturbances favoring tumor development. However, neither the extent of hypomethylation nor of retroelement reactivation are precisely known. We therefore assessed DNA methylation and expression of three major classes of retroelements (LINE-1, HERV-K and AluY in human urinary bladder cancer tissues and cell lines by pyrosequencing and quantitative reverse transcription–polymerase chain reaction, respectively. We found substantial global LINE-1 DNA hypomethylation in bladder cancer going along with a shift towards full-length LINE-1 expression. Thus, pronounced differences in LINE-1 expression were observed, which may be promoted, among others, by LINE-1 hypomethylation. Significant DNA hypomethylation was found at the HERV-K_22q11.23 proviral long terminal repeat (LTR in bladder cancer tissues but without reactivation of its expression. DNA methylation of HERVK17, essentially absent from normal urothelial cells, was elevated in cell lines from invasive bladder cancers. Accordingly, the faint expression of HERVK17 in normal urothelial cells disappeared in such cancer cell lines. Of 16 additional HERV-Ks, expression of 7 could be detected in the bladder, albeit generally at low levels. Unlike in prostate cancers, none of these showed significant expression changes in bladder cancer. In contrast, expression of the AluYb8 but not of the AluYa5 family was significantly increased in bladder cancer tissues. Collectively, our findings demonstrate a remarkable specificity of changes in expression and DNA methylation of retroelements in bladder cancer with a significantly different pattern from that

  13. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D0 or SF2). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  14. Stability of RNA and DNA in Bone Marrow Cells, Demonstrated with Tritiated Cytidine and Thymidine

    International Nuclear Information System (INIS)

    DNA and RNA metabolism was studied using tritiated thymidine (H3Th), a specific precursor for DNA, and tritiated cytidine (H3C), a common precursor for both RNA and DNA. With H3C, differential incorporation into RNA, DNA or the soluble pool was determined autoradiographically in the single cell, and/or chemically for cell populations by means of differential extraction using appropriate treatment with perchloric acid. Initial turnover studies in the Hela cell with H3C indicated the precursor role of nuclear RNA for cytoplasmic RNA. Conservation and distribution of label in the RNA fraction was consistent with major macromolecular RNA stability, and continued incorporation of label into the DNA fraction was consistent with the presence of a late precursor for DNA. Similar findings were observed in the immature bone marrow cells of the rat studied over a period of several days after intravenous administration of H3C. The amount of tritium activity in the acid-soluble' RNA and DNA fractions was followed chemically and/or autoradiographically. The three curves were found to be parallel from the first day after injection and parallel to curves for tritium label in DNA following H3Th administration. The expected rate of fall off in label, calculated from kinetics of the rat bone marrow cell populations studied separately by H3Th and autoradiography, assuming no turnover of RNA or DNA and loss of label only by loss of marrow cells by division and maturation, was in agreement with the slopes obtained. The results indicate that, once synthesized, soluble and macromolecular RNA is retained by the bone marrow cell in a manner similar to DNA. Newly formed RNA and DNA are diluted in the cells only through cell division. (author)

  15. Differential Regulation of NF-κB-Mediated Proviral and Antiviral Host Gene Expression by Primate Lentiviral Nef and Vpu Proteins

    Directory of Open Access Journals (Sweden)

    Daniel Sauter

    2015-02-01

    Full Text Available NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with β-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.

  16. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Science.gov (United States)

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker. PMID:25988180

  17. Advances in radiation biology: Radiosensitization in DNA and living cells

    Science.gov (United States)

    Lacombe, S.; Sech, C. Le

    2009-06-01

    One fundamental goal of radiation biology is the evolution of concepts and methods for the elaboration of new approaches and protocols for the treatment of cancers. In this context, the use of fast ions as ionizing particles offers the advantage of optimizing cell killing inside the tumor whilst preserving the surrounding healthy tissues. One extremely promising strategy investigated recently is the addition of radiosensitizers in the targeted tissue. The optimization of radiotherapy with fast ions implies a multidisciplinary approach to ionizing radiation effects on complex living systems, ranging from studies on single molecules to investigations of entire organisms. In this article we review recent studies on ion induced damages in simple and complex biological systems, from DNA to living cells. The specific aspect of radiosensitization induced by metallic atoms is described. As a fundamental result, the addition of sensitizing compounds with ion irradiation may improve therapeutic index in cancer therapy. In conclusion, new perspectives are proposed based on the experience and contribution of different communities including Surface Sciences, to improve the development of radiation biology.

  18. The effect of bleomycin on DNA synthesis in ataxia telangiectasia lymphoid cells

    International Nuclear Information System (INIS)

    Bleomycin, a radiomimetic glycopeptide, inhibits de novo DNA synthesis in ataxia telangiectasia lymphoblastoid B cells to a markedly lesser extent than in normal and xeroderma pigmentosum lymphoid cells. This observation is similar to that following ionizing radiation; however, the effect is slower following the chemical treatment. Recovery of the normal cells occurs 15-18 hours after treatment, whereas the ataxia telangiectasia lines do not attain normal levels of DNA synthesis during the entire 24-hour observation period. Similar differences were not observed following treatment with mitomycin C, a bifunctional alkylating agent, indicating a specific effect of bleomycin on DNA synthesis in ataxia telangiectasia cells. Following bleomycin treatment and preincubation with hydroxyurea, residual DNA synthesis in ataxia telangiectasia cells was similar to that in both normal and xeroderma pigmentosum lymphoid lines, suggesting that the capacity to repair the induced DNA lesion is present

  19. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH in buccal cells

    Directory of Open Access Journals (Sweden)

    E. I. Cortés-Gutiérrez

    2012-12-01

    Full Text Available DNA breakage detection-fluorescence in situ hybridization (DBD-FISH is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91. In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  20. Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments.

    OpenAIRE

    Fritz, G.; Tano, K.; Mitra, S.; Kaina, B

    1991-01-01

    The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA...

  1. How GSH level changes DNA-fragmentation of plasmid DNA and DNA Synthesis of tumor cells treated by Cu (TAAB)Cl2

    International Nuclear Information System (INIS)

    Rate of DNA synthesis of leukemic cell lines K562 and L1210 was monitored by incorporation of radiolabelled 3H thymidine to unsoluble TSA-cell fraction. Obtained results indicate that three days treatment with Cu(TAAB)Cl2 in various concentrations had no effect on inhibition of DNA synthesis. Changes of thiols concentrations in the presence of H2O2 and Cu(TAAB)Cl2 was determined. Oxidation of thiols in the dependence on Cu(II) ions or Cu(TAAB)Cl2 concentration was evaluated. Analysis of GSH products was performed using HPLC after pre-colon derivatization

  2. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  3. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

    DEFF Research Database (Denmark)

    Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris;

    2016-01-01

    Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for...... detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA......, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear...

  4. Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

    Science.gov (United States)

    Zhang, Pinghu; Liu, Fei; Guo, Fang; Zhao, Qiong; Chang, Jinhong; Guo, Ju-Tao

    2016-07-01

    Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted. PMID:27083116

  5. Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

    Science.gov (United States)

    Dizhe, E B; Ignatovich, I A; Burov, S V; Pohvoscheva, A V; Akifiev, B N; Efremov, A M; Perevozchikov, A P; Orlov, S V

    2006-12-01

    This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene. PMID:17223788

  6. A modified Phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients

    Directory of Open Access Journals (Sweden)

    Clemens Hufnagl

    2013-03-01

    Full Text Available Searching for new cancer biomarkers, circulating cell-free DNA (cfDNA has become an appealing target of interest as an elevated level of cfDNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since cfDNA can be isolated from the circulation and other body fluids of patients without harming their physical condition, cfDNA is becoming a promising candidate as a novel non-invasive biomarker for cancer. The challenge in the diagnostic analysis of cfDNA is its very low presence in human plasma/serum and its partially strong fragmentation. Here we evaluated a modified phenol/chloroform extraction method for the isolation of cfDNA and compared it with published standard methods for cfDNA isolation.

  7. Study on the correlation between CT appearance and nuclear DNA content in renal clear cell carcinomas

    International Nuclear Information System (INIS)

    Objective: To study the correlation of CT appearance with nuclear DNA content in renal clear cell carcinomas. Methods: Fifty-eight cases of renal clear cell carcinomas proved by surgery and pathology were examined with abdominal CT scan before operation. DNA content was determined by imaging analyzer, and DNA contents were calculated. Study on the correlation between CT appearance and nuclear DNA content was performed. Results; (1) DNA contents of tumors with diameters >5.0 cm were significantly higher than those of tumors with diameters ≤5.0 cm (t=5.860, P0.05). Conclusion: Renal clear cell carcinomas with diameters >5.0 cm, intratumoral necrosis, liquefaction, cystic degeneration, lymph nodes metastases, invasion of renal vein or inferior vena cava, invasion of adjacent organs or distant metastases had higher DNA content. Those tumors had higher malignant biological behavior

  8. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  9. Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells.

    Directory of Open Access Journals (Sweden)

    P J Hastings

    Full Text Available Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s, tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also

  10. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    Science.gov (United States)

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis.

  11. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    International Nuclear Information System (INIS)

    Highlights: ► DNA methylation is dynamic and flexible and changes rapidly upon cell activation. ► DNA methylation controls the inducible gene expression in a given cell type. ► Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  12. Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

    International Nuclear Information System (INIS)

    The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin

  13. Cell-free (RNA and cell-associated (DNA HIV-1 and postnatal transmission through breastfeeding.

    Directory of Open Access Journals (Sweden)

    James Ndirangu

    Full Text Available INTRODUCTION: Transmission through breastfeeding remains important for mother-to-child transmission (MTCT in resource-limited settings. We quantify the relationship between cell-free (RNA and cell-associated (DNA shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum. MATERIALS AND METHODS: Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission. RESULTS: There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33-4.59 vs. aHR 1.52 (95% CI, 1.17-1.96, respectively]. At 6 months, cell-free and cell-associated levels (per ml in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64-3.92 vs. 1.73 (95% CI 0.94-3.19, respectively]. CONCLUSIONS: The findings suggest that cell-associated virus level (per ml is more important for early postpartum HIV-1 transmission (at 6 weeks than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on

  14. Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ashton, Thomas M; Hickson, Ian D

    2011-01-01

    The Sgs1-Rmi1-Top3 "dissolvasome" is required for the maintenance of genome stability and has been implicated in the processing of various types of DNA structures arising during DNA replication. Previous investigations have revealed that unprocessed (X-shaped) homologous recombination repair (HRR......) intermediates persist when S-phase is perturbed by using methyl methanesulfonate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 or Top3. However, the precise nature of these persistent DNA structures remains poorly characterized. Here, we report that ectopic expression of either of two heterologous...... to RusA or GEN1(1-527), demonstrating specificity of these HJ resolvases for MMS-induced X-structures in vivo. These data suggest that the X-structures persisting in cells with impaired Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 directly promotes X-structure removal, because the...

  15. DNA damage-induced cell death: lessons from the central nervous system

    Institute of Scientific and Technical Information of China (English)

    Helena Lobo Borges; Rafael Linden; Jean YJ Wang

    2008-01-01

    DNA damage can, but does not always, induce cell death. While several pathways linking DNA damage signals to mitochondria-dependent and -independent death machineries have been elucidated, the connectivity of these pathways is subject to regulation by multiple other factors that are not well understood. We have proposed two conceptual models to explain the delayed and variable cell death response to DNA damage: integrative surveillance versus autonomous pathways. In this review, we discuss how these two models may explain the in vivo regulation of cell death induced by ionizing radiation (IR) in the developing central nervous system, where the death response is regulated by radiation dose, cell cycle status and neuronal development.

  16. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise;

    2015-01-01

    damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

  17. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma

    OpenAIRE

    Primerano, Simona; Burnelli, Roberta; Carraro, Elisa; Pillon, Marta; Elia, Caterina; Farruggia, Piero; Sala, Alessandra; Vinti, Luciana; Buffardi, Salvatore; Basso, Giuseppe; Mascarin, Maurizio; Mussolin, Lara

    2016-01-01

    Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first che...

  18. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    Directory of Open Access Journals (Sweden)

    Peixin Huang

    2015-06-01

    Full Text Available Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1 and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR, ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  19. Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

    International Nuclear Information System (INIS)

    Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism

  20. Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

    Science.gov (United States)

    Stumpf, F; Schoendube, J; Gross, A; Rath, C; Niekrawietz, S; Koltay, P; Roth, G

    2015-07-15

    Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing. PMID:25771302

  1. Efficiency, specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells

    International Nuclear Information System (INIS)

    The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke. Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region. The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases α, δ and ε. A similar extent of bypass was observed also in XP-V cells, defective in pol η, both in the absence and presence of aphidicolin. DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%). The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported. Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed. This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol η and pol δ, when present, are involved in bypass of 8-oxoguanine in vivo

  2. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    International Nuclear Information System (INIS)

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [3H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line

  3. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cell

    Science.gov (United States)

    Agasti, Sarit S.; Liong, Monty; Peterson, Vanessa M.; Lee, Hakho; Weissleder, Ralph

    2012-01-01

    DNA barcoding is an attractive technology as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here, we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells. PMID:23092113

  4. Light chain editors of anti-DNA receptors in human B cells

    OpenAIRE

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André; Weigert, Martin

    2014-01-01

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing m...

  5. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    OpenAIRE

    Ivanov, Maxim; Baranova, Ancha; Butler, Timothy; Spellman, Paul; Mileyko, Vladislav

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Result...

  6. DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Sonny C.; Crow, Ailey K.; Lam, Wilbur A.; Bertozzi, Carolyn R.; Fletcher, Daniel A.; Francis, Matthew B.

    2008-08-01

    Measurement of receptor adhesion strength requires the precise manipulation of single cells on a contact surface. To attach live cells to a moveable probe, DNA sequences complementary to strands displayed on the plasma membrane are introduced onto AFM cantilevers (see picture, bp=base pairs). The strength of the resulting linkages can be tuned by varying the length of DNA strands, allowing for controlled transport of the cells.

  7. Damage of DNA and plasma membranes in murine lymphoma cells irradiated under aerobic or hypoxic conditions

    International Nuclear Information System (INIS)

    A review of the knowledge of radiation effects on cell membranes and DNA and of repair mechanisms of radiation lesions is given. Investigations of properties of plasma membranes in L5178Y-S and L5178Y-R cells (surface charge, fluidity, transport of amino acids) indicate that there is no direct connection between membrane lesions and reproductive death. It was also found that in irradiated cells of both L5178Y-strains the rate of DNA chain elongation is the same, similarly as the amount of the initial DNA lesions and the rate of repair processes. Difference in the level of DNA synthesis inhibition is not proportional to the lethal effect. The results are also reported point to the difference between L5178Y-S and L5178Y-R cells in susceptibility of post-irradiation DNA synthesis to factors modifying chromatin conformation, such as inhibitors of (ADP-ribose)n polymerase. 221 refs. (author)

  8. DNA level in guard cells nuclei of Ornithogalum umbellatum ovary is 2C-4C

    Directory of Open Access Journals (Sweden)

    Maria Kwiatkowska

    2011-04-01

    Full Text Available The DNA content after Feulgen reaction in the guard cells and epidermis of Omithogalum umbellatum ovary was cytophotometrically measured in different phases of flower development. Only in bud of flowers guard cells DNA content was 2C while in full blown flowers it was higher, between 2C-4C. This observation was supported by autoradiographic studies with 3H-thymidine which was incorporated into guard cell nuclei in the ovary epidermis of newly developed flowers. Thus DNA level in O. umbellatum guard cells was higher than those in other plants described in literature. On the other hand, DNA content in the epidermis cells increased gradually with ovary growth reaching the maximum level of 8C in some cells.

  9. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome.

    Science.gov (United States)

    Heilbronn, R; zur Hausen, H

    1989-01-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo. Images PMID:2547992

  10. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  11. Role of Endonuclease G in Exogenous DNA Stability in HeLa Cells.

    Science.gov (United States)

    Misic, V; El-Mogy, M; Haj-Ahmad, Y

    2016-02-01

    Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts. PMID:27260396

  12. The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

    International Nuclear Information System (INIS)

    ''In vitro'' experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A1, B1, E1, E2 and Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE1 and PgE2 at 5 ng/ml and at 2,5 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE1, PgE2 and PgF2 at 2,5 μg/ml. (author)

  13. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

    Science.gov (United States)

    Nocker, Andreas; Camper, Anne K

    2006-03-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology. PMID:16517648

  14. Induction of interferon and cell death in response to cytosolic DNA in chicken macrophages.

    Science.gov (United States)

    Vitak, Nazarii; Hume, David A; Chappell, Keith J; Sester, David P; Stacey, Katryn J

    2016-06-01

    Responses to cytosolic DNA can protect against both infectious organisms and the mutagenic effect of DNA integration. Recognition of invading DNA is likely to be fundamental to eukaryotic cellular life, but has been described only in mammals. Introduction of DNA into chicken macrophages induced type I interferon mRNA via a pathway conserved with mammals, requiring the receptor cGAS and the signalling protein STING. A second pathway of cytosolic DNA recognition in mammalian macrophages, initiated by absent in melanoma 2 (AIM2), results in rapid inflammasome-mediated pyroptotic cell death. AIM2 is restricted to mammals. Nevertheless, chicken macrophages underwent lytic cell death within 15 min of DNA transfection. The mouse AIM2-mediated response requires double stranded DNA, but chicken cell death was maintained with denatured DNA. This appears to be a novel form of rapid necrotic cell death, which we propose is an ancient response rendered redundant in mammalian macrophages by the appearance of the AIM2 inflammasome. The retention of these cytosolic DNA responses through evolution, with both conserved and non-conserved mechanisms, suggests a fundamental importance in cellular defence. PMID:26828392

  15. Harnessing the p53-PUMA Axis to Overcome DNA Damage Resistance in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Xiaoguang Zhou

    2014-12-01

    Full Text Available Resistance to DNA damage–induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC. This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage–induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA. We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage–induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

  16. Elucidation of the mechanism of X-ray induced DNA duplication observed in human Gorlin cells

    International Nuclear Information System (INIS)

    A phenomenon in which DNA synthesis level increases rapidly after x-ray irradiation has found out in the cells which originate in Gorlin patients. A gene, by which an expression level changes after x-ray irradiation, is searched in the human Gorlin cells by the mRNA differential display method. The DNA synthesis level decreases in normal human cell after x-ray irradiation of 2 Gy dose, but increases twice in the Gorlin cell. Expression levels of gene SMT3A, however decrease clearly in the Gorlin cells after the irradiation. The relations between expression levels of gene SMT3M, a protein like ubichitin, and DNA synthesis levels are searched. DNA synthesis activity in normal human cells, which are treated by antisese oligonucleotide and suppressed expression of the genes SMT3A, increases after x-ray irradiation. An increase of the DNA synthesis level after the irradiation is not a phenomenon in particular cells, but indicates the possibility of general phenomena in normal human cells. It is reported that the gene SMT3A combines with a glycosylase which operates in DNA repairing process. The protein modification of gene SMT3A indicates a possibility for controlling of stress protection mechanism in the cells. (M. Suetake)

  17. Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents

    International Nuclear Information System (INIS)

    A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells). Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium. The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours. No significant DNA amplification was detectable in the treated cells

  18. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    UV (ultraviolet) damaged DNA and its repair of various cultured animal cells were observed by radioimmunoassay using anti-serum against the UV irradiation induced heat-degenerated DNA. There is some difference among the cells of used animals according to their DNA repairabilities. The cells were divided into four groups according to the existence or strength of their repairabilities. 1) excision repair type: cells of men and chimpanzees. 2) photoreactivation type: cells derived from Tachydromus tachydromoides and chicks. 3) photoreactivation with excision repair: cells of rats, kangaroos and mosquitos. 4) non-excision repair type: cells of mice, Meriones and rats. Animal cells have plural types of repair. Main types of repair will differ according to the kind of animals. (Ichikawa, K.)

  19. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    Science.gov (United States)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  20. Protective effect of deoxynucleotide triphosphates on DNA damage in different mammalian cells exposed to -radiation

    Directory of Open Access Journals (Sweden)

    Elmaghraby, T

    2002-09-01

    Full Text Available DNA is generally considered to be the most critical cellular target when considering the lethal, carcinogenic and mutagenic effects of drugs, radiation and environmental chemicals. So the study aim to the determination the damaging effect of -radiation on DNA and the protective effect of deoxynucleotide triphosphates (dNTPs. The study includes three cell types, lymphocytes, kidney cells of African gree monkey (Vero and hepatocellular carcinoma of human (HePG2 exposed to 1-5 Gy of -radiation and by using fluorometric analysis of DNA unwinding (FADU method, DNA damage was measured after radiation. The cells were divided into two groups: The first received 5x10-5 dNTPs from 0-30 minutes after radiation, while the second group was not supplemented with deoxynucleotides. Clonogenic survival for vero and HePG2 cell lines was measured. The results revealed that the increase of irradiation dose precipitates an increase of DNA strand breaks. The slope curve of initial DNA damage and mean inactivation dose (D differ between vero and HepG2 cell line by a factor of up 3.5 and 2, respectively. dNTPs have clear ameliorating effect on DNA damage. FADU method can play an important role in the choice of a suitable treatment (radiation or drugs and its dosage according to measurement of DNA damages in selective malignant tissues. Moreover, using dNTPs mixture can reduce the side effect of these treatment especially after experimentally on live mammals (mice .

  1. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    OpenAIRE

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  2. Radiation-induced DNA double-strand break frequencies in human squamous cell carcinoma cell lines of different radiation sensitivities

    International Nuclear Information System (INIS)

    DNA neutral (pH 9-6) filter elution was used to measure radiation-induced DNA double-strand break (dsb) frequencies in eight human squamous cell carcinoma cell lines with radiosensitivities (D0) ranging from 1.07 to 2.66 Gy and D-bar values ranging from 1.46 to 4.08 Gy. Elution profiles of unirradiated samples from more radiosensitive cell lines were all steeper in slope than profiles from resistant cells. The shapes of the dsb induction curves were curvilinear and there was some variability from cell line to cell line in the dose-response for the induction of DNA dsb after exposures to 5-100 Gy 60Co γ-rays. There was no relation between shapes of survival curves and shapes of the dose-responses for the induction of DNA dsb. At low doses (5-25 Gy), three out of four of the more sensitive cell lines (D-bar3.0 Gy). Although the low-dose (5-25 Gy) elution results were variable, they suggest that DNA neutral elution will detect differences between sensitive and resistant tumour cells in initial DNA dsb frequencies. (author)

  3. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    OpenAIRE

    1984-01-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmetha...

  4. Comparison of DNA loop size and super-coiled domain size in human cells

    International Nuclear Information System (INIS)

    DNA loop size and super-coiled domain size in HeLa cells were investigated by the nucleoid sedimentation technique as well as by analysis of the DNA-nuclearmatrix complex. After X-irradiation, the relaxation of super-coils was not correlated with the release of DNA from the nuclear matrix. The super-coiled domain size was much larger than the average DNA loop size. To explain the results, we propose that unwinding of super-coils in a DNA loop also causes unwinding in adjacent DNA loops in spite of existing attachment sites. Alternatively, the results may be explained by assuming that only very large DNA loops determine the sedimentation behaviour of nucleoids. (orig.)

  5. Assessing the DNA methylation status of single cells with the comet assay.

    Science.gov (United States)

    Wentzel, Johannes F; Gouws, Chrisna; Huysamen, Cristal; Dyk, Etresia van; Koekemoer, Gerhard; Pretorius, Pieter J

    2010-05-15

    The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I. PMID:20156416

  6. Detection of DNA damage induced by heavy ion irradiation in the individual cells with comet assay

    Science.gov (United States)

    Wada, S.; Natsuhori, M.; Ito, N.; Funayama, T.; Kobayashi, Y.

    2003-05-01

    Investigating the biological effects of high-LET heavy ion irradiation at low fluence is important to evaluate the risk of charged particles. Especially it is important to detect radiation damage induced by the precise number of heavy ions in the individual cells. Thus we studied the relationship between the number of ions traversing the cell and DNA damage produced by the ion irradiation. We applied comet assay to measure the DNA damage in the individual cells. Cells attached on the ion track detector CR-39 were irradiated with ion beams at TIARA, JAERI-Takasaki. After irradiation, the cells were stained with ethidium bromide and the opposite side of the CR-39 was etched. We observed that the heavy ions with higher LET values induced the heavier DNA damage. The result indicated that the amount of DNA damage induced by one particle increased with the LET values of the heavy ions.

  7. DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

    Institute of Scientific and Technical Information of China (English)

    Jilong Zheng; Xiaona Li; Di Shan; Han Zhang; Dawei Guan

    2012-01-01

    In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

  8. Proximity ligation in situ assay for monitoring the global DNA methylation in cells

    Directory of Open Access Journals (Sweden)

    Vallette François M

    2011-04-01

    Full Text Available Abstract Background DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. Results We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. Conclusion This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

  9. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  10. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xu; Ptasinska, Sylwia [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Klas, Matej [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Liu, Yueying [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Sharon Stack, M. [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 (United States)

    2013-06-10

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  11. DNA strand breaks induced by 125I in cultured human kidney cells and their repair

    International Nuclear Information System (INIS)

    125I was incorporated into the DNA of cultured human kidney cells. In intact cells, 4 single-strand breaks per decay were measured. Two SSBs per decay were found in isolated DNA. The numbers of double-strand breaks per decay in two experiments ranged from 1.4 to 2.7 depending on two different constants used in the calculations. A detailed microdosimetric analysis will be necessary to establish relationships between eV absorbed in DNA after 125I-decay and numbers of induced strand breaks. By incubation of the cells after exposure repair of SSBs could be demonstrated. This was not the case for DSBs

  12. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    International Nuclear Information System (INIS)

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  13. Changes of DNA content of tumor cell nuclei in different types of irradiation

    International Nuclear Information System (INIS)

    Comparative evaluation of the state of tumour cells in patients with larynx cancer under various types of radiation action in isoeffective doses using the method of DNA cytophotometry was made. It is shown that changes in tumour cell ploidy occur in cases of: 1) operative treatment without irradiation; 2) preoperational photon therapy; 3) preoperational photon-neutron therapy; 4) preoperational neutron therapy. In all the cases preoperational course of treatment by radiotherapy amounted to 35-40 Gy. It is shown that DNA content in nuclei, index of DNA accumulation, ploidy permit an objective evaluation of dynamics of tumour cell state after irradiation. 8 refs.; 1 fig

  14. Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

    OpenAIRE

    Hosogi,Nobuo; Hanakawa,Shiro; Watanabe,Sekiko; Oda,Takuzo

    1981-01-01

    A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated co...

  15. Contributions of DNA interstrand cross-links to aging of cells and organisms

    OpenAIRE

    Grillari, Johannes; Katinger, Hermann; Voglauer, Regina

    2007-01-01

    Impaired DNA damage repair, especially deficient transcription-coupled nucleotide excision repair, leads to segmental progeroid syndromes in human patients as well as in rodent models. Furthermore, DNA double-strand break signalling has been pinpointed as a key inducer of cellular senescence. Several recent findings suggest that another DNA repair pathway, interstrand cross-link (ICL) repair, might also contribute to cell and organism aging. Therefore, we summarize and discuss here that (i) s...

  16. Expression of adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus.

    OpenAIRE

    Watson, C J; Hay, R T

    1990-01-01

    Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus t...

  17. The influence of tumour cell DNA content on survival in colorectal cancer: a detailed analysis.

    OpenAIRE

    Armitage, N C; Ballantyne, K. C.; Evans, D F; Clarke, P; Sheffield, J.; Hardcastle, J. D.

    1990-01-01

    We have investigated the influence of tumour cell DNA content (ploidy) on survival of 416 patients undergoing excisional surgery for colorectal cancer. Two hundred and eleven (51%) tumours had an abnormal DNA content (aneuploid or tetraploid). There was no correlation between ploidy status, sex, age and pathological stage, histological grade, tumour site, local tumour extension or assessment of curability. Patients with tumours with an abnormal DNA content had a poorer survival 68/211 (32%) t...

  18. Activation of a DNA Damage Checkpoint Response in a TAF1-Defective Cell Line

    OpenAIRE

    Buchmann, Ann M.; Skaar, Jeffrey R.; DeCaprio, James A.

    2004-01-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphoryl...

  19. Changes in liver cell DNA methylation status in diabetic mice affect its FT-IR characteristics.

    Directory of Open Access Journals (Sweden)

    Benedicto de Campos Vidal

    Full Text Available Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile.The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO and adequate software.Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

  20. Inhibition of the Nedd8 system sensitizes cells to DNA Inter-strand crosslinking agents

    OpenAIRE

    Kee, Younghoon; Huang, Min; Chang, Sophia; Moreau, Lisa A.; Park, Eunmi; Smith, Peter G.; D’Andrea, Alan D.

    2012-01-01

    The Fanconi Anemia (FA) pathway is required for repair of DNA interstrand crosslinks (ICLs). FA pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as Cisplatin. Conversely, hyperactivation of the FA pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the FA pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the...

  1. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA

    OpenAIRE

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-01-01

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratical...

  2. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    OpenAIRE

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2015-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and a...

  3. Enhancement of therapeutic drug and DNA delivery into cells by electroporation* Enhancement of therapeutic drug and DNA delivery into cells by electroporation

    Science.gov (United States)

    Rabussay, Dietmar; Dev, Nagendu B.; Fewell, Jason; Smith, Louis C.; Widera, Georg; Zhang, Lei

    2003-02-01

    The effectiveness of potentially powerful therapeutics, including DNA, is often limited by their inability to permeate the cell membrane efficiently. Electroporation (EP) also referred to as `electropermeabilization' of the outer cell membrane renders this barrier temporarily permeable by inducing `pores' across the lipid bilayer. For in vivo EP, the drug or DNA is delivered into the interstitial space of the target tissue by conventional means, followed by local EP. EP pulses of micro- to millisecond duration and field strengths of 100-1500 V cm-1 generally enhance the delivery of certain chemotherapeutic drugs by three to four orders of magnitude and intracellular delivery of DNA several hundred-fold. We have used EP in clinical studies for human cancer therapy and in animals for gene therapy and DNA vaccination. Late stage squamous cell carcinomas of the head and neck were treated with intratumoural injection of bleomycin and subsequent EP. Of the 69 tumours treated, 25% disappeared completely and another 32% were reduced in volume by more than half. Residence time of bleomycin in electroporated tumours was significantly greater than in non-electroporated lesions. Histological findings and gene expression patterns after bleomycin-EP treatment indicated rapid apoptosis of the majority of tumour cells. In animals, we demonstrated the usefulness of EP for enhanced DNA delivery by achieving normalization of blood clotting times in haemophilic dogs, and by substantially increasing transgene expression in smooth muscle cells of arterial walls using a novel porous balloon EP catheter. Finally, we have found in animal experiments that the immune response to DNA vaccines can be dramatically enhanced and accelerated by EP and co-injection of micron-sized particles. We conclude that EP represents an effective, economical and safe approach to enhance the intracellular delivery, and thus potency, of important drugs and genes for therapeutic purposes. The safety and pharmaco

  4. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    International Nuclear Information System (INIS)

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas

  5. Recovery of CHO cells from hyperthermic potentiation to x rays: repair of DNA and chromatin

    International Nuclear Information System (INIS)

    Above the critical temperature, ca. 42.50C, hyperthermic potentiation of Chinese hamster ovary (CHO) cells to x irradiation was accompanied by increased binding of nonhistone proteins to DNA and by reduced rates of rejoining of DNA strand breaks. These biochemical changes were reversed as the cells recovered from the hyperthermic exposures at 370C. If the hyperthermically treated cells were incubated at 370C before x irradiation, the ratio of nonhistone protein to DNA returned to normal in 12 h but the depressed rate of rejoining of DNA strand breaks and increased cell radiosensitivity remained unaltered. Cell radiosensitivity began to decrease after 12 h and recovery from hyperthermia-potentiated radiosensitivity was complete by 48 h. In the same interval, the rate of rejoining of DNA strand breaks also returned to normal. From this behavior, we conclude that the reduction in the rate of rejoining of DNA strand breaks involved changes in DNA structure which were restored only after the thermal enhancement of protein binding was reversed. These experiments provide support for the viewpoint that critical hyperthermic potentiation (i.e., above 42.50C for CHO cells) may have logistical advantages over subcritical hyperthermic potentiation (i.e., below 42.50C) in clinical situations

  6. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Joshua W Modell

    2014-10-01

    Full Text Available Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.

  7. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Science.gov (United States)

    Modell, Joshua W; Kambara, Tracy K; Perchuk, Barrett S; Laub, Michael T

    2014-10-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage. PMID:25350732

  8. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Directory of Open Access Journals (Sweden)

    Sarah Breitbach

    Full Text Available Cell-free DNA (cfDNA in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD 20.1 (23.8 ng/ml; range 5.1-183.0 ng/ml compared to the healthy controls (9.7 (4.2 ng/ml; range 1.6-23.7 ng/ml. With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

  9. Studies of DNA damage in tumor cells induced by radiation exposure by means of single cell gel electrophoresis

    International Nuclear Information System (INIS)

    In order to detect the radiation sensitivity of tumor cells, the methods of single cell gel electrophoresis (SCGE) and 3H-TdR incorporation were applied to determine effects of radiation on single strand breaks of DNA in K562, SMMC-772 and HOS8603 cell lines. The experimental results proved that the cell migration distance was prolonged, DNA syntheses was restricted and the 3H-TdR incorporation rate was decreased in the above-mentioned 3 tumor cells lines after irradiation by 60Co γ rays (1-20 Gy)

  10. Heterogeneity in white blood cells has potential to confound DNA methylation measurements.

    Directory of Open Access Journals (Sweden)

    Bjorn T Adalsteinsson

    Full Text Available Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179 and in two white blood cell fractions (n = 20, isolated using density gradient centrifugation, in four CGIs (CpG Islands located in genes HHEX (10 CpG sites assayed, KCNJ11 (8 CpGs, KCNQ1 (4 CpGs and PM20D1 (7 CpGs. Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter explained up to 40% (p<0.0001 of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

  11. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    International Nuclear Information System (INIS)

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

  12. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    International Nuclear Information System (INIS)

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope

  13. Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    Science.gov (United States)

    Rygiel, Karolina A.; Tuppen, Helen A.; Grady, John P.; Vincent, Amy; Blakely, Emma L.; Reeve, Amy K.; Taylor, Robert W.; Picard, Martin; Miller, James; Turnbull, Doug M.

    2016-01-01

    Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but ∼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evidence of mitochondrial genome duplications allowing replication and clonal expansion of these complex rearranged molecules. The extended spectrum of mtDNA rearrangements in single cells provides insight into the process of clonal expansion which is fundamental to our understanding of the role of mtDNA mutations in ageing and disease. PMID:27131788

  14. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Malkondu, Sait [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Uyar, Pembegul [Selcuk University, Faculty of Science, Department of Biology, 42075 Konya (Turkey); Selcuk University, Advanced Technology Research and Application Center, Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey)

    2015-03-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope.

  15. DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

    International Nuclear Information System (INIS)

    We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

  16. Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

    Science.gov (United States)

    Broering, R; Trippler, M; Werner, M; Real, C I; Megger, D A; Bracht, T; Schweinsberg, V; Sitek, B; Eisenacher, M; Meyer, H E; Baba, H A; Weber, F; Hoffmann, A-C; Gerken, G; Schlaak, J F

    2016-05-01

    The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV. PMID:26833585

  17. Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells

    Institute of Scientific and Technical Information of China (English)

    BAI Jigang; L(U) Yi; BAI Qiaoling

    2007-01-01

    CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.

  18. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    Science.gov (United States)

    Yu, L. D.; Wongkham, W.; Prakrajang, K.; Sangwijit, K.; Inthanon, K.; Thongkumkoon, P.; Wanichapichart, P.; Anuntalabhochai, S.

    2013-06-01

    Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

  19. Treg cell resistance to apoptosis in DNA vaccination for experimental autoimmune encephalomyelitis treatment.

    Directory of Open Access Journals (Sweden)

    Youmin Kang

    Full Text Available BACKGROUND: Regulatory T (Treg cells can be induced with DNA vaccinations and protect mice from the development of experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis (MS. Tacrolimus (FK506 has been shown to have functions on inducing immunosuppression and augmenting apoptosis of pathologic T cells in autoimmune disease. Here we examined the therapeutic effect of DNA vaccine in conjunction with FK506 on EAE. METHODOLOGY/PRINCIPAL FINDINGS: After EAE induction, C57BL/6 mice were treated with DNA vaccine in conjunction with FK506. Functional Treg cells were induced in treated EAE mice and suppressed Th1 and Th17 cell responses. Infiltrated CD4 T cells were reduced while Treg cells were induced in spinal cords of treated EAE mice. Remarkably, the activated CD4 T cells augmented apoptosis, but the induced Treg cells resisted apoptosis in treated EAE mice, resulting in alleviation of clinical EAE severity. CONCLUSIONS/SIGNIFICANCE: DNA vaccine in conjunction with FK506 treatment ameliorates EAE by enhancing apoptosis of CD4 T cells and resisting apoptosis of induced Treg cells. Our findings implicate the potential of tolerogenic DNA vaccines for treating MS.

  20. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wongkham, W. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K.; Inthanon, K. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Wanichapichart, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Membrane Science and Technology Research Center, Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkla 90112 (Thailand); Anuntalabhochai, S. [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2013-06-15

    Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

  1. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    International Nuclear Information System (INIS)

    Low-energy ion beams at a few tens of keV were demonstrated to be able to induce exogenous macromolecules to transfer into plant and bacterial cells. In the process, the ion beam with well controlled energy and fluence bombarded living cells to cause certain degree damage in the cell envelope in nanoscales to facilitate the macromolecules such as DNA to pass through the cell envelope and enter the cell. Consequently, the technique was applied for manipulating positive improvements in the biological species. This physical DNA transfer method was highly efficient and had less risk of side-effects compared with chemical and biological methods. For better understanding of mechanisms involved in the process, a systematic study on the mechanisms was carried out. Applications of the technique were also expanded from DNA transfer in plant and bacterial cells to DNA transfection in human cancer cells potentially for the stem cell therapy purpose. Low-energy nitrogen and argon ion beams that were applied in our experiments had ranges of 100 nm or less in the cell envelope membrane which was majorly composed of polymeric cellulose. The ion beam bombardment caused chain-scission dominant damage in the polymer and electrical property changes such as increase in the impedance in the envelope membrane. These nano-modifications of the cell envelope eventually enhanced the permeability of the envelope membrane to favor the DNA transfer. The paper reports details of our research in this direction.

  2. Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk

    Science.gov (United States)

    Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. From an original sample-set of 753 male and...

  3. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  4. Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180.

    Science.gov (United States)

    Reuter, S; Delius, H; Kahn, T; Hofmann, B; zur Hausen, H; Schwarz, E

    1991-01-01

    The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs. Images PMID:1716694

  5. Interleukin-6 Promotes Tumorigenesis by Altering DNA Methylation in Oral Cancer Cells

    OpenAIRE

    Gasche, Jacqueline A; Hoffmann, Jürgen; Boland, C. Richard; Goel, Ajay

    2011-01-01

    Worldwide oral squamous cell carcinoma (OSCC) accounts for more than 100,000 deaths each year. Chronic inflammation constitutes one of the key risk factors for OSCC. Accumulating evidence suggests that aberrant DNA methylation may contribute to OSCC tumorigenesis. This study investigated whether chronic inflammation alters DNA methylation and expression of cancer-associated genes in OSCC.

  6. Comparative single cell and flow DNA analysis in aspiration biopsies from breast carcinomas.

    Science.gov (United States)

    Auer, G; Tribukait, B

    1980-11-01

    The DNA distribution patterns in fine needle aspirates from 17 breast carcinomas was analysed, using single cell and flow cytophotometric techniques. A good correlation was observed to exist between the modal DNA values obtained by the two methods. Advantages and disadvantages of the two methods are discussed. PMID:7010915

  7. Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field.

    LENUS (Irish Health Repository)

    Shields, L

    2014-10-31

    Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells are not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci\\/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant

  8. Accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with E.coli formamidopyrimidine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Mara Foresta

    Full Text Available Cigarette smoke (CS is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP. Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na(+K(+-ATPase locus (oua(r were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells' capacity to repair damaged DNA.

  9. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.)

  10. Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors

    Directory of Open Access Journals (Sweden)

    Diesch Claude

    2009-11-01

    Full Text Available Abstract Background With the aim to simplify cancer management, cancer research lately dedicated itself more and more to discover and develop non-invasive biomarkers. In this connection, circulating cell-free DNA (ccf DNA seems to be a promising candidate. Altered levels of ccf nuclear DNA (nDNA and mitochondrial DNA (mtDNA have been found in several cancer types and might have a diagnostic value. Methods Using multiplex real-time PCR we investigated the levels of ccf nDNA and mtDNA in plasma samples from patients with malignant and benign breast tumors, and from healthy controls. To evaluate the applicability of plasma ccf nDNA and mtDNA as a biomarker for distinguishing between the three study-groups we performed ROC (Receiver Operating Characteristic curve analysis. We also compared the levels of both species in the cancer group with clinicopathological parameters. Results While the levels of ccf nDNA in the cancer group were significantly higher in comparison with the benign tumor group (P P P P = 0.022. The level of ccf nDNA was also associated with tumor-size (2 cmP = 0.034. Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (cut-off: 1866 GE/ml; sensitivity: 81%; specificity: 69%; P P Conclusion Our data suggests that nuclear and mitochondrial ccf DNA have potential as biomarkers in breast tumor management. However, ccf nDNA shows greater promise regarding sensitivity and specificity.

  11. Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine

    International Nuclear Information System (INIS)

    The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of 3H-thymidine-5'-triphosphate (3H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in 3H-TTP labelling index (3H-TTP LI) was apparent together with an inhibition of 3H-leucine incorporation (3H-LEU LI) into lymphoblasts. A moderate decrease in 3H-TDR labelling index (3H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of 3H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in 3H-TTP LI, 3H-TDR LI and 3H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G1. (author)

  12. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA

    Science.gov (United States)

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M. Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-01-01

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA. PMID:27230542

  13. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    International Nuclear Information System (INIS)

    Highlights: ► We success serial SCNT through the third generation using pig fibroblasts. ► Donor-specific mtDNA in the recloned pigs was detected. ► SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor’s mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  14. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of); Shim, Hosup [Department of Physiology, Dankook University School of Medicine, Cheonan 330 714 (Korea, Republic of); Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June [Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Suwon (Korea, Republic of); Kim, Jin-Hoi [Department of Animal Biotechnology, Konkuk University, Seoul 143 701 (Korea, Republic of); Lee, Jeong Woong, E-mail: jwlee@kribb.re.kr [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  15. Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells.

    Science.gov (United States)

    Groehler, Arnold; Villalta, Peter W; Campbell, Colin; Tretyakova, Natalia

    2016-02-15

    N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage. PMID:26692166

  16. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    Science.gov (United States)

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-01-01

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis. PMID:27188728

  17. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    Science.gov (United States)

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Results In this study, we investigated the association between epigenetic landscapes of human tissues evident in the patterns of cfDNA in plasma by deep sequencing of human cfDNA samples. We have demonstrated that baseline characteristics of cfDNA fragmentation pattern are in concordance with the ones corresponding to cell lines-derived. To identify the loci differentially represented in cfDNA fragment, we mapped the transcription start sites within the sequenced cfDNA fragments and tested for association of these genomic coordinates with the relative strength and the patterns of gene expressions. Preselected sets of house-keeping and tissue specific genes were used as models for actively expressed and silenced genes. Developed measure of gene regulation was able to differentiate these two sets based on sequencing coverage near gene transcription start site. Conclusion Experimental outcomes suggest that cfDNA retains characteristics previously noted in genome-wide analysis of chromatin structure, in particular, in MNase-seq assays. Thus far the analysis of the DNA fragmentation pattern may aid further developing of cfDNA based biomarkers for a variety of human conditions. PMID:26693644

  18. Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The G1 cells of the diploid yeast Saccharomyces cerevislae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium. In the present paper, it has been shown that S. cerevislae cells γ-irradiated in the G1 phase of cell cycle are capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 deg C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, ''fast'' phase is completed within 30-40 min; wheras the second, ''slow'' one, within 48 h. Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not practically influence this process. It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S, cerevislae are separate pathways of DNA DSB repair in yeast

  19. Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

    OpenAIRE

    Underhill, T M; Flintoff, W F

    1989-01-01

    A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated g...

  20. Sorafenib attenuates p21 in kidney cancer cells and augments cell death in combination with DNA-damaging chemotherapy

    OpenAIRE

    Inoue, Hiromi; Hwang, Sung Hee; Wecksler, Aaron T.; Hammock, Bruce D.; Robert H. Weiss

    2011-01-01

    There are few effective therapeutic options for metastatic renal cell carcinoma (RCC). Conventional chemotherapeutic agents are ineffective since these tumors are unusually resistant to DNA damage, likely due to an exuberant DNA repair response. Sorafenib, as one of the few available effective therapeutic options for metastatic RCC, has been shown to inhibit cell proliferation by inhibition of tyrosine kinases. We have recently shown that sorafenib inhibits soluble epoxide hydrolase, which ca...

  1. Bleomycin - induced DNA damage and DNA repair in chicken embryo cells as compared to X-irradiation

    International Nuclear Information System (INIS)

    Following in vitro- and in ovo-exposure of chicken embryo cells, the level of bleomycin (BM) - induced damage was evaluated by using DNA synthesis, nucleoid sedimentation (SED), and viscometry of alkaline cell lysates (VISC). This damage was compared to X-irradiation, using 5.9-378 nM BM in vitro, 1.5-116 μg BM/egg in ovo, and 2-32 Gy, respectively, in vitro as well as in ovo. With respect to BM, the most notable result is the increase in DNA synthesis and VISC at the lowest concentrations of the drug. A decrease in both parameters was observed at high BM concentrations and following exposure to X-rays, concomitantly with an increase in SED. Regarding the radiomimetic drug BM and X-rays, different modes of DNA damage and DNA repair are suggested by previous investigations and the present results. Therefore, further evidence is presented, that the chicken embryo can act as a simple, rapid and inexpensive test system to characterize the biological effects of many nucleo- and/or cytotoxic agents. (orig.)

  2. Dendritic cell targeted liposomes–protamine–DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine

    International Nuclear Information System (INIS)

    To construct mannosylated liposomes/protamine/DNA (LPD) carriers for DNA vaccine targeting to dendritic cells (DCs), a mannosylated cholesterol derivative (Man-C6-Chol) was synthesized via simple ester linkage and amide bonds. Then, the Man-C6-Chol was applied to LPD formulation as a synthetic ligand. The physicochemical properties of mannosylated LPD (Man-LPD) were first evaluated, including the size and zeta potential, morphology and the ability to protect DNA against DNase I degradation. Man-LPD showed a small size with a stable viral-like structure. In comparison to non-mannose liposomes/LPD (Man-free liposomes/LPD), mannosylated liposomes/LPD (Man-liposomes/Man-LPD) exhibited higher efficiency in both intracellular uptake (2.3-fold) and transfection (4.5-fold) in vitro. Subsequent MTT assays indicated that the LPD carriers had low toxicity on the tested cells. Afterwards, the investigation into the maturation activation on primary bone marrow-derived DCs (BMDCs) showed that both Man-LPD and Man-free LPD induced remarkable up-regulation of CD80, CD86 and CD40 on BMDCs. Inspired by these studies, we can conclude that the synthetic mannosylated LPD targeting to DCs was a potential carrier for DNA vaccine. (paper)

  3. Dendritic cell targeted liposomes-protamine-DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine

    Science.gov (United States)

    Li, Pan; Chen, Simu; Jiang, Yuhong; Jiang, Jiayu; Zhang, Zhirong; Sun, Xun

    2013-07-01

    To construct mannosylated liposomes/protamine/DNA (LPD) carriers for DNA vaccine targeting to dendritic cells (DCs), a mannosylated cholesterol derivative (Man-C6-Chol) was synthesized via simple ester linkage and amide bonds. Then, the Man-C6-Chol was applied to LPD formulation as a synthetic ligand. The physicochemical properties of mannosylated LPD (Man-LPD) were first evaluated, including the size and zeta potential, morphology and the ability to protect DNA against DNase I degradation. Man-LPD showed a small size with a stable viral-like structure. In comparison to non-mannose liposomes/LPD (Man-free liposomes/LPD), mannosylated liposomes/LPD (Man-liposomes/Man-LPD) exhibited higher efficiency in both intracellular uptake (2.3-fold) and transfection (4.5-fold) in vitro. Subsequent MTT assays indicated that the LPD carriers had low toxicity on the tested cells. Afterwards, the investigation into the maturation activation on primary bone marrow-derived DCs (BMDCs) showed that both Man-LPD and Man-free LPD induced remarkable up-regulation of CD80, CD86 and CD40 on BMDCs. Inspired by these studies, we can conclude that the synthetic mannosylated LPD targeting to DCs was a potential carrier for DNA vaccine.

  4. Potent T cell Responses Induced by Single DNA Vaccine Boosted with Recombinant Vaccinia Vaccine

    Institute of Scientific and Technical Information of China (English)

    Lianxing Liu; Chao Qiu; Yang Huang; Jianqing Xu; Yiming Shao

    2013-01-01

    Plasmid DNA,an effective vaccine vector,can induce both cellular and humoral immune responses.However,plasmid DNA raises issues concerning potential genomic integration after injection.This issue should be considered in preclinical studies.Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China.This virus has also been considered as a successful vaccine vector against a few infectious diseases.Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine.To develop a safer immunization strategy,a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice.Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity,preferential T cell Vβ receptor usage and high sensitivity to anti-CD3 antibody stimulation.No differences in T cell responses were observed among one,two or three DNA prime/rTV boost regimens.This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.

  5. Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

  6. DNA-content and synthesis rate in human melanoma cells in vitro after hyperthermia and radiation

    International Nuclear Information System (INIS)

    Human melanoma cells were cultured over 168 hours. DNA-content and 3H-thymidine incorporation were measured after 800 R and hyperthermia of 400C and 440C for 3 and 6 hours. An early and a late effect could be distinguished. 3 and 6 hours after irradiation alone no alteration of DNA-synthesis was observed. After heat treatment at 400C for 6 hours the DNA-synthesis was increased immediately. 420C and 440C deminished the rate of DNA-synthesis. The combined treatment (heat and irradiation) suppressed the overshooting rate of DNA-synthesis. Accordingly after heat treatment at 400C for 6 hours the DNA-content was higher than the controls and the 3 hours-400C treated cultures measured over a period of 48 hours. Thereafter the DNA-content showed little or no alterations compared with the controls. The heat treatment at 440C reduced the DNA-content heavily, followed by a relative increase at 120 hours. The DNA-synthesis rate showed the same effect. The combined treatment suppressed this late increase. However, 24 hours after combined treatment the incorporation of thymidine into the DNA was higher at 440C-6 than 440C-3 hours, 40C-3 hours, than 400C-6 hours, although the DNA-content was very low. The results show synergistic effects of hyperthermia and radiation on the DNA-synthesis and content if one considers the effects at the later periods (120-168 hours). However, a stimulating effect is found on the DNA-synthesis if the melanoma cells are incubated at 400C for 6 hours. (orig.)

  7. Change and Significance of Mitochondrial DNA Copy Number in Esophageal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zongwen Liu; Zhihua Zhao; Qiumin Zhao; Shenglei Li; Dongling Gao; Xia Pang; Kuisheng Chen; Yunhan Zhang

    2007-01-01

    OBJECTIVE To compare the differences of mitochondrial DNA (mtDNA)copies among the tissues of esophageal squamous cell carcinoma (ESCC),para-neoplastic tissue and normal mucous membrane of the esophagus,and to study the relationship between the mtDNA and the occurrence and development of esophageal squamous cell carcinoma.METHODS The mtDNA copies of 42 specimens with the ESCC,paraneoplastic mucous tissue and normal mucous membrane of the esophagus were determined using real-time fluorescence quantitative PCR.The mtDNA was analyzed using agarose gel electrophoresis.RESULTS The mtDNA from all of the tissues (42/42) from the ESCC,para-neoplastic tissue and normal esophageal mucous membranes was analyzed.showing thal there were an average mtDNA copy number of 27.1894x106 μg DNA.9.4102x106 μg DNA and 5.9347x106 μg DNA,from the respective tissues.There were significant differences (F=27.83,P<0.05) in mtDNA copy number among the three.A positive band was shown at 403 bp after qel electrophoresis of the PCR products.and the lane where the ESCC mtDNA located was rather bright.which was in accordance with the result of the real-time PCR determination.CONCLUSION An increase in the mtDNA copy number is related to the occurrence and development of ESCC.

  8. DNA synthesis as an index of the cell reaction to irradiation and other damaging exposures

    International Nuclear Information System (INIS)

    Recent investigation results, showing the outlook of DNA synthesis suppresion determination method as a test for estimating and predicting cell sensitivity to irradiation and other damageing exposures are presented. Advantages of such a method are noted

  9. Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery

    CERN Document Server

    Escoffre, Jean-Michel; Favard, Cyril; Teissié, Justin; Dean, David S; Rols, Marie-Pierre

    2011-01-01

    Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2 ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is propos...

  10. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Goto, Yamafumi [Department of Dermatology, Shinshu University School of Medicine, Matsumoto (Japan); Takata, Minoru [Department of Dermatology, Okayama University Graduate School of Medical Dentistry and Pharmaceutical Sciences, Okayama (Japan); Turkson, James; Li, Xiaoman Shawn [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Zervos, Antonis S., E-mail: azervos@mail.ucf.edu [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States)

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  11. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    International Nuclear Information System (INIS)

    Research highlights: → THAP5 is a DNA-binding protein and a transcriptional repressor. → THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. → THAP5 induction correlates with the degree of apoptosis in melanoma cell population. → THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  12. Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage

    DEFF Research Database (Denmark)

    Skovgaard, T; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    2010-01-01

    enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP...

  13. Free electron laser irradiation at 200 microns affects DNA synthesis in living cells

    International Nuclear Information System (INIS)

    We describe the effect of a 200-microns wavelength free electron laser beam on the ability of asynchronized and synchronized mammalian tissue culture cells to incorporate tritiated thymidine. Compared to controls (unexposed cells), a significant proportion of exposed cells exhibited a reduction in isotope incorporation. The results suggest that this wavelength may affect DNA synthesis

  14. Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk

    OpenAIRE

    Smith, Alyson J.; Nelson, Natalie G.; Oommen, Saji; Hartjes, Katherine A.; Folmes, Clifford D.; Terzic, Andre; Nelson, Timothy J.

    2012-01-01

    The pluripotent cell-purging assay validated herein demonstrates that pluripotent cells are selectively hypersensitive to DNA damage-induced apoptosis as a function of the specific apoptotic inducer protein Puma. Risk of dysregulated growth is decreased and the safety profile of transplant-ready, bioengineered progenitor cells is augmented.

  15. Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

    OpenAIRE

    Nagy, E.

    1992-01-01

    A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the dete...

  16. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Müller Mathias

    2007-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

  17. Capacity of ultraviolet-induced DNA repair in human glioma cells

    International Nuclear Information System (INIS)

    A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment. (author)

  18. Single-Cell Analysis of Ribonucleotide Reductase Transcriptional and Translational Response to DNA Damage

    OpenAIRE

    Mazumder, Aprotim; Tummler, Katja; Bathe, Mark; Samson, Leona D.

    2013-01-01

    The ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronized Saccharomyces cerevisiae cells suggest that RNR mRNA production is maximal in late G1 and S phases; however, damaged DNA induces RNR transcription throughout the cell cycle. But such en masse measurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and p...

  19. Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

    Directory of Open Access Journals (Sweden)

    István Fűri

    Full Text Available Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin, DNA methyltransferase 3a (DNMT3a and NFκB (for treated HDFα cells.Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05 mRNA level alteration in 118 genes (logFc≥1, p≤0.05, including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A, metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1, tumor biomarker (CEACAM5, metabolic genes (i.e. INSIG1, LIPG, messenger molecule genes (i.e. DAPP, CREB3L2. Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05, including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β, STING pathway (ADAR, IRF7, CXCL10, CASP1 and the FGF2 gene.DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling

  20. Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

    International Nuclear Information System (INIS)

    Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose-effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D0 value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D0 values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile site was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as open-quotes cometsclose quotes by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. 37 refs., 2 figs., 1 tab

  1. A DNA-Fullerene Conjugate as a Template for Supramolecular Chromophore Assemblies: Towards DNA-Based Solar Cells.

    Science.gov (United States)

    Ensslen, Philipp; Gärtner, Stefan; Glaser, Konstantin; Colsmann, Alexander; Wagenknecht, Hans-Achim

    2016-01-01

    A fullerene was covalently attached to a (dA)20 template that serves as structural scaffold to self-assemble an ordered and mixed array of ethynyl-pyrene- and ethynyl-Nile-red-nucleoside conjugates. Fluorescence spectroscopy revealed evidence for energy transfer between the two different chromophores. Moreover, fluorescence quenching is significantly enhanced by the attached fullerene in mixed assemblies of different chromophore ratios. This indicates exciton dissociation by electron transfer from the photo-generated exciton on the chromophore stack to the fullerene. The fullerene-DNA-conjugate was integrated as a photo-active layer in solar cells that showed charge-carrier generation in the spectral regime of all three components of the conjugate. This work clearly demonstrates that DNA is suitable as structural element for chromophore assemblies in future organic optoelectronic devices, such as solar cells. PMID:26689149

  2. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    OpenAIRE

    Seok-Jo Kim; Paul Cheresh; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidenc...

  3. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency

    OpenAIRE

    Qiang Tu; Jia Yin; Jun Fu; Jennifer Herrmann; Yuezhong Li; Yulong Yin; Francis Stewart, A.; Rolf Müller; Youming Zhang

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and re...

  4. Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNA.

    OpenAIRE

    Bowman, E. H.; Pathak, V K; Hu, W S

    1996-01-01

    A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA ...

  5. TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

    Science.gov (United States)

    Meller, Stephan; Domizio, Jeremy Di; Voo, Kui S; Friedrich, Heike C; Chamilos, Georgios; Ganguly, Dipyaman; Conrad, Curdin; Gregorio, Josh; Roy, Didier Le; Roger, Thierry; Ladbury, John E; Homey, Bernhard; Watowich, Stanley; Modlin, Robert L; Kontoyiannis, Dimitrios P; Liu, Yong-Jun; Arold, Stefan T; Gilliet, Michel

    2016-01-01

    Interleukin 17–producing helper T cells (TH17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human TH17 cell–derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, TH17 cell–derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26–DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of TH17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death. PMID:26168081

  6. HIV-1 latency in actively dividing human T cell lines

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2008-04-01

    Full Text Available Abstract Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.

  7. A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from γ-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. Only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining), thought to be due to the increased accessibility of SQ-9G DNA in vitro. (author)

  8. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Directory of Open Access Journals (Sweden)

    Goli eSamimi

    2015-04-01

    Full Text Available A range of molecular alterations found in tumor cells, such as DNA mutations and methylation changes, is also reflected in cell-free circulating DNA (circDNA released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. DNA methylation is a common molecular alteration found in many cancer types. Unlike DNA mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. DNA methylation is reflected within circDNA and therefore detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

  9. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Heath Murray

    2014-10-01

    Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  10. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2016-01-01

    Full Text Available Background. Noninvasive prenatal screening (NIPS is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

  11. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Science.gov (United States)

    Cao, Yang; Hoppman, Nicole L.; Kerr, Sarah E.; Sattler, Christopher A.; Borowski, Kristi S.; Wick, Myra J.; Highsmith, W. Edward; Aypar, Umut

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA) screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information. PMID:26998368

  12. Molecular behavior of DNA in a cell-sized compartment coated by lipids

    CERN Document Server

    Hamada, T; Shimobayashi, S F; Ichikawa, M; Takagi, M

    2015-01-01

    The behavior of long DNA molecules in a cell-sized confined space was investigated. We prepared water-in-oil droplets covered by phospholipids, which mimic the inner space of a cell, following the encapsulation of DNA molecules with unfolded coil and folded globule conformations. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space. Globular DNA showed a cell-size-dependent unfolding transition after adsorption on the membrane. Furthermore, when DNA interacted with a two-phase membrane surface, DNA selectively adsorbed on the membrane phase, such as an ordered or disordered phase, depending on its conformation. We discuss the mechanism of these trends by considering the free energy of DNA together with a polyamine in the solution. The free energy of our model was consistent with the present experimental data. The cooperative interaction of DNA and polyamines with a membrane surface leads to the size-dependent behavior of molec...

  13. Determination of the Action Spectrum of UVR-Induced Mitochondrial DNA Damage in Human Skin Cells.

    Science.gov (United States)

    Latimer, Jennifer A; Lloyd, James J; Diffey, Brian L; Matts, Paul J; Birch-Machin, Mark A

    2015-10-01

    Biological responses of human skin to UVR including cancer and aging are largely wavelength-dependent, as shown by the action spectra of UVR-induced erythema and nuclear DNA (nDNA) damage. A molecular dosimeter of UVR exposure is therefore required. Although mitochondrial DNA (mtDNA) damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin, its wavelength dependency is unknown. The current study solves this problem by determining the action spectrum of UVR-induced mtDNA damage in human skin. Human neonatal dermal fibroblasts and primary human adult keratinocyte cells were irradiated with increasing doses of UVR. Dose-response curves of mtDNA damage were produced for each of the UVR sources and cell types, and an action spectrum for each cell type was determined by mathematical induction. Similarities between these mtDNA damage action spectra and previously determined nDNA damage were observed, with the most detrimental effects occurring over the shorter UVR wavelengths. Notably, a statistically significant (P300 nm, possibly indicating a wider picture of depth dependence in sensitivity. This finding has implications for disease/photodamage mechanisms and interventions. PMID:26030182

  14. High recovery of cell-free methylated DNA based on a rapid bisulfite-treatment protocol

    Directory of Open Access Journals (Sweden)

    Pedersen Inge

    2012-03-01

    Full Text Available Abstract Background Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amounts of cell-free DNA in plasma, analytical sensitivity is of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulfite-mediated deamination of cytosine. Cytosine is rapidly converted to uracil during bisulfite treatment, whereas 5-methylcytosine is only slowly converted. Hence, bisulfite treatment converts an epigenetic modification into a difference in sequence, amenable to analysis either by sequencing or PCR based methods. However, the recovery of bisulfite-converted DNA is very poor. Results Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours. Conclusions The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.

  15. 60Co-γ-irradiation of dried DNA and isolated cell nuclei of chicken erythrocytes

    International Nuclear Information System (INIS)

    In this work low molecular products, which resulted from γ-irradiation of dried DNA, were isolated and quantitatively determined. Unchanged nucleic bases were released. The irradiation was successful in a vacuum as well as in oxygen. In the case of the drily irradiated DNA, the base release made up 30% of the total strand breaks. The release of DNA bases was also first studied in cell nuclei of eucaryotic cells, the erythrocyte nuclei of chicken blood. The second part of this work dealt with the isolation and identification of radiation induced changes in the 2-dioxyribose unit of DNA. In the third part it was investigated, whether as a result of γ-irradiation of DNA malonic dialdehyde was formed. It could be shown that neither malonic aldehyde nor basic propenal were formed, but instead products, which were still bound to the DNA and formed a chromophore with 2-thio barbituric acid. In this work the direct irradiation effect was investigated in DNA systems as well as in erythrocyte nuclei. By the isolation of low molecular irradiation products this work offers a contribution to the understanding of the irradiation chemistry in an eucaryotic cell, in which the direct effect on the DNA and the resulting radiation damage were given a deciding role for genetic information. (orig./MG)

  16. Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Simon, Perikles

    2012-07-01

    The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis

  17. Purification by DNA affinity precipitation of the cellular factors HEB1-p67 and HEB1-p94 which bind specifically to the human T-cell leukemia virus type-I 21 bp enhancer.

    OpenAIRE

    Lombard-Platet, G; Jalinot, P

    1993-01-01

    Transcription driven by the proviral promoter of the Human T-cell Leukemia Virus type I (HTLV-I) is tightly regulated by the Tax1 transactivator. This viral protein potently induces the enhancer activity of a 21 bp motif repeated three times in the promoter. We have previously shown that this induction results from the binding of Tax1 to this enhancer sequence and that this association is mediated by the cellular factor HEB1. In this paper we report the purification of this factor by chromato...

  18. Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

    OpenAIRE

    Schmidt, Nora; Hennig, Thomas; Remigiusz A Serwa; Marchetti, Magda; O'Hare, Peter

    2015-01-01

    ABSTRACT Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cy...

  19. Radioadapted chicken embryo cells: challenge specificity and alterations in higher-order DNA structure

    International Nuclear Information System (INIS)

    Radioadapted chicken embryo cells (X-irradiation in ovo with 10 cGy at the 14th day of development with priming periods of 24 h) were treated in vitro by challenge doses of 14 different DNA- and/or chromatin-interactive agents, including X-rays. A decrease in the cellular damage, as measured by scheduled DNA synthesis, was only observed with X-irradiation. Sedimentation of nucleoids as well as viscosity of alkaline lysates from ethidium bromide (0.35-400 μg/ml)-, vovobiocin (125-1800 μg/ml)-, and hyperthermia (30 min at 43 and 45 )-treated cells suggest a higher tendency of radioadapted cells to undergo positive DNA supercoiling. When DNA from adapted and non-adapted chicken embryo cells was used as substrate, neither its digestion by DNase I nor the inhibition of DNase I activity by various DNA-interactive agents was changed in primed cells. From the previous investigations as well as from the present results it is concluded that an increase of tightening of protein-DNA interactions within the nuclear matrix is a molecular determinant of the elevated radiation resistance in radioadapted chicken embryo cells. (orig.)

  20. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    Science.gov (United States)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.