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Sample records for cell proviral dna

  1. Damaging the Integrated HIV Proviral DNA with TALENs.

    Directory of Open Access Journals (Sweden)

    Christy L Strong

    Full Text Available HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs to target a highly conserved sequence in the transactivation response element (TAR of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

  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. 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...

  6. 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...

  7. 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.

  8. 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...... 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 inherently more likely...

  9. Detection of Bovine Leukaemia Virus Antibodies and Proviral DNA in Colostrum Replacers.

    Science.gov (United States)

    Choudhury, B; Finnegan, C; Phillips, A; Horigan, M; Pollard, T; Steinbach, F

    2015-10-01

    Great Britain has been bovine leukaemia virus (BLV) disease free since 1999. We recently reported three separate incidents of BLV seropositivity on farms with home-reared cattle due to the use of colostrum replacer rather than infection with BLV (Emerg. Infect. Dis., 19, 2013, 1027). These cases were all linked via the use of the same brand of colostrum replacer. Here, we investigate further by examining multiple brands of colostrum replacer for proviral DNA and BLV antibodies. BLV antibodies were detected in 7 of the colostrum replacers tested, with PCR concurring in two cases. Thus, the use of these BLV antibody-positive colostrum replacers may also lead to false-positive serological diagnostics. PMID:24268042

  10. Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.

    Science.gov (United States)

    Jangam, Sujit R; Yamada, Douglas H; McFall, Sally M; Kelso, David M

    2009-08-01

    PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 microl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%. PMID:19644129

  11. 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...... 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 inherently more likely...

  12. 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

  13. 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

  14. 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 ...

  15. Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia.

    Science.gov (United States)

    Seu, Lillian; Mwape, Innocent; Guffey, M Bradford

    2014-07-01

    The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5' and 3' region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5' and 3' proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes.

  16. 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

  17. 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 ...

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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...

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

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    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.

  2. 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

  3. 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.

  4. 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

  5. Analysis of HIV-1 proviral DNA in peripheral blood cells from long-term HAART-treated AIDS patients in China%经长期HAART治疗的中国HIV-AIDS患者外周血细胞内HIV-1前病毒DNA的检测及其意义

    Institute of Scientific and Technical Information of China (English)

    陈霞; 姚运海; 郑煜煌; Diallo MA; 何艳; 周华英; 谌资; 罗艳; 贺波; 贺梅

    2012-01-01

    Objective The aim of this study was to verify that NK cells were one type of the virus reservoir cells,and to observe whether NK cells,T lymphocytes and monocyte are long-term viral reservoir cells of HIV-1.Methods A group of 15 chronic HIV-l-infected adults,who received 4 ~7 years-HAART treatment[ mean(62.45 ± 13.53) months],participated in this study for crosssectional study.After the ethical approval was obtained,a volume (40 ml) of peripheral whole blood was obtained from each patient.The number of T cell subgroup was detected with flow cytomctry.The NK cells,T lymphocytes,and monocytes were separated from peripheral blood mononuclear cells (PBMC) using microbead sorting method ( CD3 +,CD14 + and CD56 + antibody microbeads).DNA was extracted with human DNA kit.Real-time fluorescent quantitative PCR was used to detect the serum HIV RNA and DNA in the three types of cells.Graphpad prism5 software was used to analyze the collected data.Results Among 15 patients with 4 ~ 7-year-HAART treatment,plasma HIV-1 RNA of all the patients are undetectable; however,HIV-1 DNA was detected in 6 patients'T lymphocyte [ mean(2.03 ± 0.48)logl0copies/106 cells],in 4 patients'Nk cells [mean ( 1.82 ± 0.32 ) logl0copies/106 cells ],and in 2 patients'monocytes [ mean( 1.75 ± 0.19) logl0copies/106 cells ].Conclusions These findings revealed that NK cells were another important HIV cellular reservoir besides T-lymphocytes and monocytes,and T-lymphocytes might be the main long-lasting HIV reservoir.%目的 探索NK细胞是否是HIV-1病毒的储存库细胞之一,并观察在长期HAART治疗后,T淋巴细胞及单核细胞内是否仍有HIV-1前病毒DNA存在.方法 对15例规范接受HAART治疗4~7年的HIV-AIDS患者采集血标本进行横断面研究.使用磁珠分选法从外周血单个核细胞(PBMC)中分离出T淋巴细胞、单核细胞和NK细胞,通过HIV实时荧光定量检测外周血HIV-1 RNA病毒载量和NK细胞、单核细胞、T淋巴细胞内的HIV-1

  6. 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.

  7. Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas.

    Science.gov (United States)

    Kim, Rachel; Trubetskoy, Alla; Suzuki, Takeshi; Jenkins, Nancy A; Copeland, Neal G; Lenz, Jack

    2003-02-01

    The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (Fos, Gfi1, Lef1, Myb, Myc, Runx3, and Sox3), all three D cyclins, Ras signaling pathway components (Rras2/TC21 and Rasgrp1), and Cmkbr7/CCR7. The most frequent target was Rras2. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of Rras2, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated Rras2 contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.

  8. 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...

  9. 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.

  10. 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.

  11. 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

  12. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

    Science.gov (United States)

    Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

    2016-01-01

    We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

  13. Pim2 is important for regulating DNA damage response in multiple myeloma cells

    Science.gov (United States)

    Ramachandran, J; Santo, L; Siu, K T; Panaroni, C; Raje, N

    2016-01-01

    Pan proviral integrations of Moloney virus (PIM) inhibition in multiple myeloma (MM) results in reduced cell viability in tested human-derived MM cell lines and reduces tumor burden in xenograft mouse models, making PIMs important therapeutic targets for the disease. PIM kinase inhibitors are currently being tested clinically in MM. We sought to elucidate the role of the various PIMs in MM. Our data demonstrate that Pim2 has a significant role in MM cell cytotoxicity. Our data provide evidence for a novel role for Pim2 in the regulation of the DNA damage response (DDR). Knockdown of Pim2 upregulates several downstream DDR markers, mimicking the effects of doxorubicin (Dox) treatment of MM cells, and suggesting a role for the kinase as a negative regulator of this pathway. Dox-induced DNA damage results in a decrease in Pim2 levels, placing the kinase directly downstream of the site of Dox-DNA binding. Overexpression of Pim2 confers a slight survival advantage against Dox through antiapoptotic activity, further underscoring its relevance in the DDR pathway. These data provide insights into a novel mechanism of PIM kinase activity and provide the framework for designing therapeutic approaches in MM. PMID:27564460

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

    Institute of Scientific and Technical Information of China (English)

    WANG; Yu; CUI; Zhizhong; JIANG; Shijin

    2006-01-01

    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.

  15. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Hirotaka Ebina

    Full Text Available DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 endonuclease system targeting HIV long terminal repeats (LTR. Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  16. 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 ...

  17. 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...

  18. DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Siew Ching Ngai

    2015-01-01

    Full Text Available Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP reporter gene driven by cytomegalovirus (CMV promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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...

  5. DNA damage response in adult stem cells.

    Science.gov (United States)

    Insinga, Alessandra; Cicalese, Angelo; Pelicci, Pier Giuseppe

    2014-04-01

    This review discusses the processes of DNA-damage-response and DNA-damage repair in stem and progenitor cells of several tissues. The long life-span of stem cells suggests that they may respond differently to DNA damage than their downstream progeny and, indeed, studies have begun to elucidate the unique stem cell response mechanisms to DNA damage. Because the DNA damage responses in stem cells and progenitor cells are distinctly different, stem and progenitor cells should be considered as two different entities from this point of view. Hematopoietic and mammary stem cells display a unique DNA-damage response, which involves active inhibition of apoptosis, entry into the cell-cycle, symmetric division, partial DNA repair and maintenance of self-renewal. Each of these biological events depends on the up-regulation of the cell-cycle inhibitor p21. Moreover, inhibition of apoptosis and symmetric stem cell division are the consequence of the down-regulation of the tumor suppressor p53, as a direct result of p21 up-regulation. A deeper understanding of these processes is required before these findings can be translated into human anti-aging and anti-cancer therapies. One needs to clarify and dissect the pathways that control p21 regulation in normal and cancer stem cells and define (a) how p21 blocks p53 functions in stem cells and (b) how p21 promotes DNA repair in stem cells. Is this effect dependent on p21s ability to inhibit p53? Such molecular knowledge may pave the way to methods for maintaining short-term tissue reconstitution while retaining long-term cellular and genomic integrity.

  6. 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

  7. Telomere Length, Proviral Load and Neurologic Impairment in HTLV-1 and HTLV-2-Infected Subjects

    Directory of Open Access Journals (Sweden)

    Benjamin Usadi

    2016-08-01

    Full Text Available Short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that analysis of telomere length (TL in human T-cell lymphotropic virus (HTLV infection and HTLV-associated neuropathy might provide clues to the etiology of HTLV-associated disease and viral dynamics. A subset of 45 human T-cell lymphotropic virus type 1 (HTLV-1, 45 human T-cell lymphotropic virus type 2 (HTLV-2, and 45 seronegative subjects was selected from the larger HTLV Outcomes Study (HOST cohort, matched on age, sex and race/ethnicity. Telomere-to-single-copy gene (T/S ratio (a measure of TL and HTLV-1 and HTLV-2 proviral loads were measured in peripheral blood mononuclear cells (PBMCs using quantitative PCR (qPCR. Vibration sensation measured by tuning fork during neurologic examinations performed as part of the HOST study allowed for an assessment of peripheral neuropathy. TL was compared between groups using t-tests, linear and logistic regression. Mean T/S ratio was 1.02 ± 0.16 in HTLV-1, 1.03 ± 0.17 in HTLV-2 and 0.99 ± 0.18 in HTLV seronegative subjects (p = 0.322. TL was not associated with HTLV-1 or -2 proviral load. Shorter TL was significantly associated with impaired vibration sense in the HTLV-2 positive group only. Overall, we found no evidence that telomere length was affected by chronic HTLV-1 and HTLV-2 infection. That TL was only associated with peripheral neuropathy in the HTLV-2-positive group is intriguing, but should be interpreted cautiously. Studies with larger sample size and telomere length measurement in lymphocyte subsets may clarify the relationship between TL and HTLV-infection.

  8. 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

  9. Existence of proviral porcine endogenous retrovirus in fresh and decellularised porcine tissues

    Directory of Open Access Journals (Sweden)

    Prabha S

    2008-01-01

    Full Text Available Purpose: Swine are expected to be utilized as xenograft donors for both whole organ and cellular transplantation. A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV. Tissue-engineered or decellularised heart valves have already been implanted in humans and have been marketed by certain companies after Food and Drug Administration (FDA approval. The aim of this study was to examine the existence of porcine endogenous retrovirus (PERV in fresh and decellularised porcine tissues. Methods: Porcine tissues (both fresh and decellularised were analysed using validated assays specific for PERV: polymerase chain reaction (PCR, reverse transcriptase polymerase chain reaction (RT-PCR. Results: PERV specific GAG sequences were found in the porcine heart tissue samples using PCR for DNA and RT- PCR for RNA. All tissue samples (both fresh and treated tissues like aortic valve, pulmonary valve and heart muscle showed the presence of PERV DNA. RT PCR for PERV was positive in all fresh tissues and was found to be negative in decellularised treated tissues. Conclusions: PCR is a rapid, specific test for the detection of PERV virus in xenografts. These findings have demonstrated that the presence of proviral DNA form of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

  10. 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...

  11. 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)

  12. 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....

  13. 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.

  14. 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

  15. 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)

    王柳; 童光志; 刘红全; 杨志彪; 仇华吉; 孔宪刚; 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.

  16. 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

  17. 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

  18. 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 ...

  19. Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

    Science.gov (United States)

    Kempster, S; Collins, M E; Brownlie, J

    2002-03-01

    The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

  20. 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

  1. 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

  2. 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...

  3. 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

    2016-06-07

    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.

  4. 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

  5. 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)

  6. DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation.

    Science.gov (United States)

    Fernández-Nestosa, María José; Monturus, Estefanía; Sánchez, Zunilda; Torres, Francisco S; Fernández, Agustín F; Fraga, Mario F; Hernández, Pablo; Schvartzman, Jorge B; Krimer, Dora B

    2013-01-01

    In mice, the proviral integration of the Friend Spleen Focus Forming Virus (SFFV) within the PU.1 locus of erythroid precursors results in the development of erythroleukemia. SFFV integrates several kilobases upstream of the PU.1 transcription initiation start site leading to the constitutive activation of the gene which in turn results in a block of erythroid differentiation. In this study we have mapped and sequenced the exact location of the retroviral integration site. We have shown that SFFV integrates downstream of a previously described upstream regulatory element (URE), precisely 2,976 bp downstream of the URE-distal element. We have also found that SFFV persists integrated within the same location in resistant cell lines that have lost their differentiation capacity and in which case PU.1 remains silent. We have examined the methylation status of PU.1 and found that in resistant cells the nearby CpG islands remained methylated in contrast to a non-methylated status of the parental cell lines. Treatment with 5-aza-2'-deoxycytidine caused resistant cells to differentiate yet only when combined with HMBA. Altogether these results strongly suggest that methylation plays a crucial role with regard to PU.1 silencing. However, although demethylation is required, it is not sufficient to overcome the differentiation impasse. We have also showed that activation blockage of the Epo/Epo-R pathway remains despite of the absence of PU.1.

  7. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

  8. 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...

  9. 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

  10. DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

    DEFF Research Database (Denmark)

    Shaknovich, Rita; Cerchietti, Leandro; Tsikitas, Lucas;

    2011-01-01

    cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably...

  11. 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.

  12. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  13. 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...

  14. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    Science.gov (United States)

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  15. 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...

  16. 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

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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...

  4. Dicer is associated with ribosomal DNA chromatin in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Lasse Sinkkonen

    Full Text Available BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/- ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

  5. 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...

  6. 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)

  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. 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

  9. Targeting DNA vaccines to myeloid cells using a small peptide.

    Science.gov (United States)

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

  10. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

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

    Science.gov (United States)

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; 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 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. PMID:26981554

  12. 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.

  13. 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...

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

    DEFF Research Database (Denmark)

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

    and 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......Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools......-synthase. As a result of this the rho0 cells have much lower ATP levels than rho+ cells. In order to mimic the ATP situation in rho0 cells the rho+ cells are incubated with the ATP synthase inhibitor, oligomycin. Similar to the rho0 cells the oligomycin incubated rho+ cells display destabilized dNTP pools upon UV...

  15. 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...

  16. 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 diver......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...

  17. 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...

  18. 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...

  19. DNA repair in human xeroderma pigmentosum and chinese hamster cells

    NARCIS (Netherlands)

    Zelle, B.

    1980-01-01

    An important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as 4-nitroquinoline-1-oxide (4N

  20. DNA repair in human xeroderma pigmentosum and Chinese hamster cells

    NARCIS (Netherlands)

    B. Zelle (Bauke)

    1980-01-01

    textabstractAn important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as 4-nitroquinoline

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

    DEFF Research Database (Denmark)

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

    and 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......Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... of this the rho0 cells have much lower ATP levels than rho+ cells. In order to mimic the ATP situation in rho0 cells the rho+ cells are incubated with the ATP synthase inhibitor, oligomycin. Similar to the rho0 cells the oligomycin incubated rho+ cells display increased dNTP pools upon UV irradiation. Our data...

  2. 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

  3. 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.

  4. 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

  5. Cell-free DNA screening and sex chromosome aneuploidies.

    Science.gov (United States)

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.

  6. 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...

  7. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Matsukura, T.; Kanda, T.; Furuno, A.; Yoshikawa, H.; Kawana, T.; Yoshiike, K.

    1986-06-01

    The authors have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1284 and 4471 (numbering system from K. Seedorf, G. Kraemmer, M. Duerst, S. Suhai, and W.G. Roewkamp), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1137 and 1138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Photoenzyme probes of photodamage to cells and cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B. M.

    1979-01-01

    Development of photoenzyme probes for detection of ultraviolet damage to cells and DNA is reviewed with special emphasis on a process using polyethylene glycol to induce cell fusion. Polyethylene glycol is easy to obtain and handle, is gentle to the cells and does not induce latent or productive virus infection; therefore, it may be a general method for insertion of exogenous enzymes into mammalian cells. (PCS)

  14. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    Science.gov (United States)

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  15. 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

  16. 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...

  17. 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

  18. 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

  19. 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)

  20. 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)

  1. 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)

  2. 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

  3. 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.

  4. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  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. [Profiles of DNA methylation in normal and cancer cells].

    Science.gov (United States)

    Weber, Michaël

    2008-01-01

    In eukaryotes, the epigenetic mark DNA methylation is found exclusively at cytosine residues in the CpG islands of genes, transposons and intergenic DNA. Among functional roles, DNA methylation is essential for mammalian embryonic development, and is classically thought to function by stably silencing promoter activity. However, until recently, understanding of the distribution of cytosine methylation in the whole genome - and hence, identification of its targets - was very limited. High-throughput methodologies, including methylated DNA immunoprecipitation, have recently revealed genome-wide mapping of DNA methylation, and provided new and unexpected data. Clearly DNA methylation is selectively associated with some key promoters- and is not a prerequisite for promoter inactivation, since strong CpG island promoters are mostly unmethylated, even when inactive. Most germline-specific genes are methylated and permanently silenced in somatic cells, suggesting a role of this mark in maintaining somatic cellular identity. These large scale studies will also help understanding the deregulation of DNA methylation associated with cancer, among which unmethylation of germinal cells genes, and recent observtion of large hypomethylated regions in tumoral specimens. The next challenge will be to understand if these methylation changes occur randomly, or more likely are specified by oncogenes or linked to environmental pressure. PMID:18789220

  7. Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1984-06-01

    Full Text Available In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97% by aphidicolin at 10 micrograms/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30% and 90% depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90% at 100 micrograms/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 micrograms/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase alpha and a non-alpha DNA polymerase (possibly DNA polymerase beta, are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase alpha in UDS favored DNA synthesis in the intranucleosomal region.

  8. [Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes].

    Science.gov (United States)

    Chumakov, M I

    2001-01-01

    Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins. PMID:11236737

  9. 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

  10. 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

  11. 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

  12. 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...

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  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. 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 cell death in Pb(NO₃)₂-treated cells, indicative of membrane 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 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

    Science.gov (United States)

    Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

    2016-10-01

    The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

  10. 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

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

    Science.gov (United States)

    Han, Bingshe; Li, Wenhao; Chen, Zuozhou; 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

  12. 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.

  13. 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

  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. 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

  16. 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.

  17. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))

    1987-06-01

    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.

  18. 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

  19. 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

  20. 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.)

  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. 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

  3. Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

    NARCIS (Netherlands)

    D. Chadha; C.J. Cornelisse; A. Schabert (A.)

    1990-01-01

    textabstractAbstract The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or near-dip

  4. Involvement of proliferating cell nuclear antigen (Cyclin) in DNA replication in living cells

    Energy Technology Data Exchange (ETDEWEB)

    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. The authors 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 replication in living cells. This result further implies that DNA polymerase /delta/ is necessary for plasmid replication in vivo, Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase /alpha/. Anti-DNA polymerase /alpha/ alone inhibited plasmid replication by 63%. Thus, DNA ploymerase /alpha/ is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase /alpha/ antibody blocked 73% of replication. They concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase /delta/. In addition, they obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymearse /alpha/, that the structure of DNA polymerase /alpha/ holoenzyme for chromosome replication is significantly different from that for plasmid replication.

  5. 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...

  6. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  7. 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.)

  8. 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

    by 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......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...... 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...

  9. Cell-free circulating tumor DNA in cancer

    Institute of Scientific and Technical Information of China (English)

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

    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 under-lying difculties 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.

  10. 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

  11. 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.

  12. 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

  13. 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...

  14. Analysis of human T-cell lymphotropic virus in CD25+ anaplastic large cell lymphoma in children.

    Science.gov (United States)

    Gualco, Gabriela; Chioato, Lucimara; Weiss, Lawrence M; Harrington, William J; Bacchi, Carlos E

    2009-07-01

    Anaplastic large cell lymphoma (ALCL) is recognized as 2 distinct diseases: anaplastic lymphoma kinase (ALK)+ ALCL and ALK- ALCL. ALK+ ALCL occurs in younger patients and has a better prognosis. Human T-cell lymphotropic virus (HTLV-1) is linked to the development of adult T-cell leukemia/lymphoma (ATLL), which frequently expresses CD25. CD25 is significantly expressed in childhood ALCL. In Brazil, HTLV-1 infection is endemic, and vertical transmission is responsible for spread to children. Of HTLV-1 carriers, 90% or more remain asymptomatic. Some cases of adult HTLV-1-related lymphomas have characteristics of ALCL but are considered CD30+ ATLL subtypes. No similar cases have been described in children. We analyzed 33 cases of pediatric ALCL, CD25+ and CD25-, for proviral HTLV-1 DNA. All cases corresponded to the common histologic ALCL type and were CD30+ in virtually all neoplastic cells. ALK expression was observed in 31 (94%) of 33 cases; CD25 was positive in 27 (82%), including 1 ALK- ALCL case. There was a strong positive correlation between ALK and CD25 expression. None of the cases showed proviral HTLV-1 DNA. ALCL in children has no relationship with HTLV-1; the frequent CD25 expression must be explained by a mechanism different from that in ATLL.

  15. 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.

  16. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different pha

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Enara eAguirre

    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.

  5. 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

  6. 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.

  7. 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

  8. 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 ...

  9. 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 ...

  10. 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 ...

  11. 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 ...

  12. 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...

  13. 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...

  14. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA

    OpenAIRE

    Qiuqiang Gao; Liang-Chun Liou; Qun Ren; Xiaoming Bao; Zhaojie Zhang

    2015-01-01

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ0). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ0 c...

  15. 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...

  16. Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells.

    Science.gov (United States)

    Ramos, Alice A; Azqueta, Amaya; Pereira-Wilson, Cristina; Collins, Andrew R

    2010-06-23

    DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study evaluated the effects of the water extracts of three Salvia sp., Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and of the major phenolic constituents, rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G), on DNA protection in Caco-2 and HeLa cells exposed to oxidative agents and on DNA repair in Caco-2 cells. The comet assay was used to measure DNA damage and repair capacity. The final concentration of each sage extract was 50 microg/mL, and concentrations of RA and L-7-G were 50 and 20 microM, respectively. After a short incubation (2 h), L-7-G protected DNA in Caco-2 cells from damage induced by H(2)O(2) (75 microM); also, after a long incubation (24 h), SF, RA, and L-7-G had protective effects in Caco-2 cells. In HeLa cells, SO, SF, and RA protected against damage induced by H(2)O(2) after 24 h of incubation. Assays of DNA repair show that SO, SF, and L-7-G increased the rate of DNA repair (rejoining of strand breaks) in Caco-2 cells treated with H(2)O(2). The incision activity of a Caco-2 cell extract on a DNA substrate containing specific damage (8-oxoGua) was also measured to evaluate effects on base excision repair (BER) activity. Preincubation for 24 h with SO and L-7-G had a BER inductive effect, increasing incision activity in Caco-2 cells. In conclusion, SO, SF, and the isolated compounds (RA and L-7-G) demonstrated chemopreventive activity by protecting cells against oxidative DNA damage and stimulating DNA repair (SO, SF, and L-7-G). PMID:20486687

  17. 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 ...

  18. Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

    OpenAIRE

    Horton, J K; Srivastava, D K; Zmudzka, B Z; Wilson, S H

    1995-01-01

    Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-...

  19. 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.

  20. 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.

  1. 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.

  2. 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...

  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. 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.

  5. 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

  6. Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

    Science.gov (United States)

    Lee, W; Johnson, J; Gough, D J; Donoghue, J; Cagnone, G L M; Vaghjiani, V; Brown, K A; Johns, T G; St John, J C

    2015-02-26

    Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

  7. 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

  8. 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

  9. 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.

  10. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines.

    Science.gov (United States)

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I (2) statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating "hub genes" - heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  11. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    Science.gov (United States)

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility.

  12. 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.

  13. 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

  14. 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

  15. Identification of tissue-specific cell death using methylation patterns of circulating DNA

    Science.gov (United States)

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-01-01

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

  16. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    Science.gov (United States)

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

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  4. File list: Oth.Unc.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.50.DNA-RNA_hybrids.AllCell.bed ...

  5. 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)

  6. 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.

  7. 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

  8. 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...

  9. 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.

  10. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  11. 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.

  12. 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

  13. 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...

  14. 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)

  15. 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

  16. Single-Cell DNA Methylome Sequencing and Bioinformatic Inference of Epigenomic Cell-State Dynamics

    Directory of Open Access Journals (Sweden)

    Matthias Farlik

    2015-03-01

    Full Text Available Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS assay that enables DNA methylation mapping in very small cell populations (μWGBS and single cells (scWGBS. Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples.

  17. 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.

  18. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    Energy Technology Data Exchange (ETDEWEB)

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  19. 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.

  20. 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

  1. 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

  2. Production of anti-double-stranded DNA antibodies in activated lymphocyte derived DNA induced lupus model was dependent on CD4+ T cells.

    Science.gov (United States)

    Wen, Z; Xu, L; Xu, W; Xiong, S

    2012-04-01

    Our previous study demonstrated that activated lymphocyte derived DNA (ALD-DNA) could function as an autoantigen to induce production of anti-double-stranded DNA (anti-dsDNA) antibodies in syngeneic BALB/c mice. Here we carefully evaluated the potential role of T cells in the induction of anti-dsDNA antibody. We demonstrated that ALD-DNA could effectively induce production of anti-dsDNA antibodies in vivo and in vitro. In contrast, ALD-DNA could not induce the generation of anti-dsDNA antibodies in nude mice. We further showed that in vivo depletion of CD3(+) T cells blocked the induction of anti-dsDNA antibodies in BALB/c mice. Notably, we demonstrated that CD4(+) but not CD8(+) T cells conferred ALD-DNA to induce anti-dsDNA antibodies. Finally, we demonstrated that adoptive transfer of CD4(+) T cells could rescue ALD-DNA induced anti-dsDNA antibodies in nude mice. Our results suggested that T helper cells were required for ALD-DNA to induce anti-dsDNA antibodies. These findings could further our understanding about the immunogenic properties of DNA and throw new light on SLE pathogenesis.

  3. 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.

  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. 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

    Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected...... 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...... patients (P cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during...

  6. DNA asymmetry in stem cells - immortal or mortal?

    Science.gov (United States)

    Yadlapalli, Swathi; Yamashita, Yukiko M

    2013-09-15

    The immortal strand hypothesis proposes that stem cells retain a template copy of genomic DNA (i.e. an 'immortal strand') to avoid replication-induced mutations. An alternative hypothesis suggests that certain cells segregate sister chromatids non-randomly to transmit distinct epigenetic information. However, this area of research has been highly controversial, with conflicting data even from the same cell types. Moreover, historically, the same term of 'non-random sister chromatid segregation' or 'biased sister chromatid segregation' has been used to indicate distinct biological processes, generating a confusion in the biological significance and potential mechanism of each phenomenon. Here, we discuss the models of non-random sister chromatid segregation, and we explore the strengths and limitations of the various techniques and experimental model systems used to study this question. We also describe our recent study on Drosophila male germline stem cells, where sister chromatids of X and Y chromosomes are segregated non-randomly during cell division. We aim to integrate the existing evidence to speculate on the underlying mechanisms and biological relevance of this long-standing observation on non-random sister chromatid segregation.

  7. 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.

  8. The DNA methylome of human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Yingrui Li

    Full Text Available 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 that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE. These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

  9. 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

  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. Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo.

    Science.gov (United States)

    Simonetti, Francesco R; Sobolewski, Michele D; Fyne, Elizabeth; Shao, Wei; Spindler, Jonathan; Hattori, Junko; Anderson, Elizabeth M; Watters, Sarah A; Hill, Shawn; Wu, Xiaolin; Wells, David; Su, Li; Luke, Brian T; Halvas, Elias K; Besson, Guillaume; Penrose, Kerri J; Yang, Zhiming; Kwan, Richard W; Van Waes, Carter; Uldrick, Thomas; Citrin, Deborah E; Kovacs, Joseph; Polis, Michael A; Rehm, Catherine A; Gorelick, Robert; Piatak, Michael; Keele, Brandon F; Kearney, Mary F; Coffin, John M; Hughes, Stephen H; Mellors, John W; Maldarelli, Frank

    2016-02-16

    Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1.

  13. 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...... of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure...... and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential. Environ. Mol. Mutagen., 2014. © 2014 Wiley Periodicals, Inc....

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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)

  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. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    Science.gov (United States)

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-06-24

    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.

  1. 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.

  2. Cytotoxicity and DNA damage associated with pyrazoloacridine in MCF-7 breast cancer cells.

    Science.gov (United States)

    Grem, J L; Politi, P M; Berg, S L; Benchekroun, N M; Patel, M; Balis, F M; Sinha, B K; Dahut, W; Allegra, C J

    1996-06-28

    We examined the effects of pyrazoloacridine (PZA), an investigational anticancer agent in clinical trials, on cytotoxicity, DNA synthesis, and DNA damage in MCF-7 human breast carcinoma cells. With PZA concentrations ranging from 0.5 to 50 microM for durations of 3-72 hr, cytotoxicity increased in proportion to the total PZA exposure (concentration x time). Inhibition of DNA and RNA syntheses increased with increasing PZA concentration x time (microM.hr). A 24-hr exposure to 1 and 10 microM PZA reduced DNA synthesis to 62 and 5% of control, respectively, decreased the proportion of cells in S phase with accumulation of cells in G2 + M phase, and inhibited cell growth at 72 hr by 68 and 100%. Newly synthesized DNA was more susceptible to damage during PZA exposure, with subsequent induction of parental DNA damage. Significant damage to newly synthesized DNA as monitored by alkaline elution was evident after a 3-hr exposure to > or = 5 microM PZA. Longer PZA exposures (> or = 10 microM for 16 hr) were required to elicit damage to parental DNA. Induction of single-strand breaks in parental DNA correlated closely with induction of double-strand breaks and detachment of cells from the monolayer. PZA-mediated DNA fragmentation was not accompanied by the generation of oligonucleosomal laddering in MCF-7 cells, but induction of very high molecular weight DNA fragmentation (0.5 to 1 Mb) was detected by pulsed-field gel electrophoresis. In vitro binding of PZA to linear duplex DNA (1 kb DNA ladder) and closed, circular plasmid DNA was demonstrated by a shift in migration during agarose electrophoresis. PZA interfered with topoisomerase I- and II-mediated relaxation of plasmid DNA in a cell-free system, but the cytotoxic effects of PZA did not appear to involve a direct interaction with topoisomerase I or II (stabilization of the topoisomerase I- or II-DNA cleavable complex). PZA-mediated cytotoxicity correlated strongly with inhibition of DNA and RNA syntheses, and damage to

  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. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  5. 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.

  6. 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....

  7. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective.

    Science.gov (United States)

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaître, Jean-Marc; De Vos, John

    2013-04-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that "genome-friendly" reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors

    Science.gov (United States)

    Ilardi, Gennaro; Acunzo, Mario; Nigita, Giovanni; Sasdelli, Federica; Celetti, Angela; Strambi, Angela; Staibano, Stefania; Croce, Carlo Maria; Chiariello, Mario

    2016-01-01

    Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest. To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase. In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a “stress support” autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors. PMID:26988910

  13. 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)

  14. 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.

  15. 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...

  16. 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.

  17. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    Science.gov (United States)

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  18. 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

  19. 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

  20. 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

  1. Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

    Science.gov (United States)

    Barwick, Benjamin G; Scharer, Christopher D; Bally, Alexander P R; Boss, Jeremy M

    2016-10-01

    The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

  2. 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.

  3. Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage.

    Science.gov (United States)

    Finzel, Ana; Grybowski, Andrea; Strasen, Jette; Cristiano, Elena; Loewer, Alexander

    2016-08-01

    A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner. PMID:27280387

  4. 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.

  5. 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

  6. 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...

  7. 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...

  8. Regenerative capacity of old muscle stem cells declines without significant accumulation of DNA damage.

    Directory of Open Access Journals (Sweden)

    Wendy Cousin

    Full Text Available The performance of adult stem cells is crucial for tissue homeostasis but their regenerative capacity declines with age, leading to failure of multiple organs. In skeletal muscle this failure is manifested by the loss of functional tissue, the accumulation of fibrosis, and reduced satellite cell-mediated myogenesis in response to injury. While recent studies have shown that changes in the composition of the satellite cell niche are at least in part responsible for the impaired function observed with aging, little is known about the effects of aging on the intrinsic properties of satellite cells. For instance, their ability to repair DNA damage and the effects of a potential accumulation of DNA double strand breaks (DSBs on their regenerative performance remain unclear. This work demonstrates that old muscle stem cells display no significant accumulation of DNA DSBs when compared to those of young, as assayed after cell isolation and in tissue sections, either in uninjured muscle or at multiple time points after injury. Additionally, there is no significant difference in the expression of DNA DSB repair proteins or globally assayed DNA damage response genes, suggesting that not only DNA DSBs, but also other types of DNA damage, do not significantly mark aged muscle stem cells. Satellite cells from DNA DSB-repair-deficient SCID mice do have an unsurprisingly higher level of innate DNA DSBs and a weakened recovery from gamma-radiation-induced DNA damage. Interestingly, they are as myogenic in vitro and in vivo as satellite cells from young wild type mice, suggesting that the inefficiency in DNA DSB repair does not directly correlate with the ability to regenerate muscle after injury. Overall, our findings suggest that a DNA DSB-repair deficiency is unlikely to be a key factor in the decline in muscle regeneration observed upon aging.

  9. 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.

  10. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@thep-center.org [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); Sangwijit, K. [Molecular Biology Laboratory, 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); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, 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); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [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); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2014-05-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  11. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

    Science.gov (United States)

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-04-02

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

  12. 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

  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. 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)...

  15. Single-cell analysis of intercellular heteroplasmy of mtDNA in Leber hereditary optic neuropathy

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Y.; Sharpe, H.; Brown, N.

    1994-07-01

    The authors have investigated the distribution of mutant mtDNA molecules in single cells from a patient with Leber hereditary optic neuropathy (LHON). LHON is a maternally inherited disease that is characterized by a sudden-onset bilateral loss of central vision, which typically occurs in early adulthood. More than 50% of all LHON patients carry an mtDNA mutation at nucleotide position 11778. This nucleotide change converts a highly conserved arginine residue to histidine at codon 340 in the NADH-ubiquinone oxidoreductase subunit 4 (ND4) gene of mtDNA. In the present study, the authors used PCR amplification of mtDNA from lymphocytes to investigate mtDNA heteroplasmy at the single-cell level in a LHON patient. They found that most cells were either homoplasmic normal or homoplasmic mutant at nucleotide position 11778. Some (16%) cells contained both mutant and normal mtDNA.

  16. 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

  17. 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

  18. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 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 As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-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 As2O3 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 As2O3 in APL treatment.

  19. 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.)

  20. 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...

  1. 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

  2. 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 ...

  3. Photoreactivation of pyrimidine dimers in DNA from thyroid cells of the teleost, Poecilia formosa

    Energy Technology Data Exchange (ETDEWEB)

    Achey, P.M.; Woodhead, A.D.; Setlow, R.B.

    1979-01-01

    We have developed and used a simple technique to estimate the quantity of pyrimidine dimers in unlabeled cellular DNA. DNA is extracted from cells, treated with an endonuclease specific for dimers, and its molecular weight estimated by its electrophoretic mobility on alkaline agarose slab gels. The technique is used to show that cells from thyroid tissue of the fish Poecilia formosa have photoreactivating activity towards dimmers in the cellular DNA.

  4. 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

    2011-11-13

    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.

  5. 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 ...

  6. HERV-K and LINE-1 DNA methylation and reexpression in urothelial carcinoma

    Directory of Open Access Journals (Sweden)

    Ulrike eKreimer

    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

  7. Gibberellic Acid enhancement of DNA turnover in barley aleurone cells.

    Science.gov (United States)

    Taiz, L; Starks, J E

    1977-08-01

    When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [(3)H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile.The buoyant density on CsCl density gradients of hormone-treated aleurone DNA is identical with that of DNA extracted from whole seedlings. After density-labeling halfseed DNA with 5-bromodeoxyuridine, a bimodal absorption profile is obtained in neutral CsCl. The light band (1.70 g/ml) corresponds to unsubstituted DNA, while the heavy band (1.725-1.74 g/ml) corresponds to a hybrid density-labeled species. GA increases the relative amount of the heavy (hybrid) peak in halfseed aleurone layer DNA, further suggesting that the hormone enhances semiconservative replication in halfseeds.DNA methylation was also demonstrated. Over 60% of the radioactivity from [(3)H-Me]methionine is incorporated into 5-methylcytosine. GA has no effect on the percentage distribution of label among the bases.It was concluded that GA enhances the rate of DNA degradation and DNA synthesis (turnover) in halfseeds, but primarily DNA degradation in isolated aleurone layers. Incorporation by isolated aleurone layers is due to DNA repair. Semiconservative replication apparently plays no physiological role in the hormone response, since both isolated aleurone layers and gamma-irradiated halfseeds respond normally. The hypothesis was advanced that endoreduplication and DNA degradation are means by which the seed stores and mobilizes deoxyribonucleotides for the embryo during

  8. DNA damage induction and tumour cell radiosensitivity : PFGE and halo measurements

    NARCIS (Netherlands)

    Woudstra, EC; Driessen, C; Konings, AWT; Kampinga, HH

    1998-01-01

    Purpose: To test whether induction of DNA damage is correlated with tumour-cell radiosensitivity. Materials and methods: Initial DNA damage caused by X-irradiation was measured in ten human tumour cell lines, which largely differed in radiosensitivity, using either the pulsed-field gel electrophores

  9. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it po

  10. Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation.

    Science.gov (United States)

    Fath, A; Bethke, P C; Jones, R L

    1999-11-01

    Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.

  11. 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...

  12. DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells.

    Science.gov (United States)

    Björkman, Andrea; Du, Likun; Felgentreff, Kerstin; Rosner, Cornelia; Pankaj Kamdar, Radhika; Kokaraki, Georgia; Matsumoto, Yoshihisa; Davies, E Graham; van der Burg, Mirjam; Notarangelo, Luigi D; Hammarström, Lennart; Pan-Hammarström, Qiang

    2015-12-15

    Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene-diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo-switched B cells from two DNA-PKcs-deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs-deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes. PMID:26546606

  13. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization

    DEFF Research Database (Denmark)

    Reinhardt, H Christian; Hasskamp, Pia; Schmedding, Ingolf;

    2010-01-01

    Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1...... expression as part of the DNA damage response in cancer cells....

  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. 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...

  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. Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Koury, M.J.; Bondurant, M.C. (Vanderbilt Univ. Medical Center, Nashville, TN (USA) Veterans Administration Medical Center, Nashville, TN (USA))

    1990-04-20

    The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with ({sup 3}H) thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.

  18. 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.

  19. 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

    , 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......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...

  20. Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Jitesh Neupane

    2015-11-01

    Full Text Available Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002. These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

  1. Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Neupane, Jitesh; Ghimire, Sabitri; Vandewoestyne, Mado; Lu, Yuechao; Gerris, Jan; Van Coster, Rudy; Deroo, Tom; Deforce, Dieter; Vansteelandt, Stijn; De Sutter, Petra; Heindryckx, Björn

    2015-11-17

    Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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...

  7. 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.

  8. 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.

  9. 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...

  10. 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...

  11. 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

  12. 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.

  13. 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

  14. 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...

  15. 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

  16. Cells by design: a mini-review of targeting cell engineering using DNA microarrays.

    Science.gov (United States)

    Jaluria, Pratik; Chu, Chia; Betenbaugh, Michael; Shiloach, Joseph

    2008-06-01

    Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes. PMID:18327555

  17. Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells.

    Science.gov (United States)

    Guo, Ju-Tao; Zhou, Tianlun; Guo, Haitao; Block, Timothy M

    2007-12-01

    Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.

  18. Simple Laboratory methods to measure cell proliferation using DNA synthesis property

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2007-01-01

    Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

  19. 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)

  20. Cell-Free Fetal DNA Testing for Prenatal Diagnosis.

    Science.gov (United States)

    Drury, S; Hill, M; Chitty, L S

    2016-01-01

    Prenatal diagnosis and screening have undergone rapid development in recent years, with advances in molecular technology driving the change. Noninvasive prenatal testing (NIPT) for Down syndrome as a highly sensitive screening test is now available worldwide through the commercial sector with many countries moving toward implementation into their publically funded maternity systems. Noninvasive prenatal diagnosis (NIPD) can now be performed for definitive diagnosis of some recessive and X-linked conditions, rather than just paternally inherited dominant and de novo conditions. NIPD/T offers pregnant couples greater choice during their pregnancy as these safer methods avoid the risk of miscarriage associated with invasive testing. As the cost of sequencing falls and technology develops further, there may well be potential for whole exome and whole genome sequencing of the unborn fetus using cell-free DNA in the maternal plasma. How such assays can or should be implemented into the clinical setting remain an area of significant debate, but it is clear that the progress made to date for safer prenatal testing has been welcomed by expectant couples and their healthcare professionals. PMID:27645814

  1. 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

  2. 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.

  3. 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.)

  4. 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...

  5. 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

  6. 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.

  7. Optimal Arrangement of Four Short DNA Strands for Delivery of Immunostimulatory Nucleic Acids to Immune Cells.

    Science.gov (United States)

    Ohtsuki, Shozo; Matsuzaki, Noriyuki; Mohri, Kohta; Endo, Masayuki; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Takahashi, Yuki; Ishiyama, Kenichi; Kadowaki, Norimitsu; Takakura, Yoshinobu; Nishikawa, Makiya

    2015-10-01

    Nanosized DNA assemblies are useful for delivering immunostimulatory cytosine-phosphate-guanine (CpG) DNA to immune cells, but little is known about the optimal structure for such delivery. In this study, we designed three different DNA nanostructures using four 55-mer oligodeoxynucleotides (ODNs), that is, tetrapod-like structured DNA (tetrapodna), tetrahedral DNA (tetrahedron), and tetragonal DNA (tetragon), and compared their potencies. Electrophoresis showed that tetrapodna was obtained with high yield and purity, whereas tetrahedron formed multimers at high ODN concentrations. Atomic force microscopy revealed that all preparations were properly constructed under optimal conditions. The thermal stability of tetrapodna was higher than those of the others. Dynamic light scattering analysis showed that all of the assemblies were about 8 nm in diameter. Upon addition to mouse macrophage-like RAW264.7 cells, tetrahedron was most efficiently taken up by the cells. Then, a CpG DNA, a ligand for toll-like receptor 9, was linked to these DNA nanostructures and added to RAW264.7 cells. CpG tetrahedron induced the largest amount of tumor necrosis factor-α, followed by CpG tetrapodna. Similar results were obtained using human peripheral blood mononuclear cells. Taken together, these results indicate that tetrapodna is the best assembly with the highest yield and high immunostimulatory activity, and tetrahedron can be another useful assembly for cellular delivery if its preparation yield is improved.

  8. Biochips for cell biology by combined dip-pen nanolithography and DNA-directed protein immobilization.

    Science.gov (United States)

    Arrabito, Giuseppe; Reisewitz, Stephanie; Dehmelt, Leif; Bastiaens, Philippe I; Pignataro, Bruno; Schroeder, Hendrik; Niemeyer, Christof M

    2013-12-20

    A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA-directed immobilization (DDI) of oligonucleotide-tagged proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA-streptavidin (DNA-STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA-STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.

  9. 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...

  10. The cell-free fetal DNA fraction in maternal blood decreases after physical activity

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Hatt, Lotte; Bach, Cathrine;

    2014-01-01

    OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has an eff...... prenatal diagnosis based on the fetal fraction, physical activity prior to sampling should be avoided.......OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has...... of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p 

  11. Immune cell activation from multivalent interactions with liquid-crystalline polycation-DNA complexes

    Science.gov (United States)

    Schmidt, Nathan; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Frasca, Loredana; Dobnikar, Jure; Frenkel, Daan; Gilliet, Michel; Wong, Gerard

    2014-03-01

    Microbial DNA can trigger type I interferon (IFN) production in plasmacytoid cells (pDCs) by binding to endosomal toll-like receptor 9 (TLR9). TLR9 in pDCs do not normally respond to self-DNA, but in certain autoimmune diseases self-DNA can complex with the polycationic antimicrobial peptide LL37 into condensed structures which allow DNA to access endosomal compartments and stimulate TLR9 in pDCs. We use x-ray studies and cell measurements of IFN secretion by pDCs to show that a broad range of polycation-DNA complexes stimulate pDCs and elucidate the criterion for high IFN production. Furthermore, we show via experiments and computer simulations that the distinguishing factor for why certain complexes activate pDCs while others do not is the self-assembled structure of the liquid-crystalline polycation-DNA complex.

  12. Characterizing the dynamical accumulation of nuclear DNA in the sperm cells of Lycium barbarum L.

    Directory of Open Access Journals (Sweden)

    Hua Deng

    2016-02-01

    Full Text Available When sperm cells of the plant Lycium barbarum L. (L. barbarum form in a style they begin to synthesize nuclear DNA (nDNA, which monotonically increases over time. To characterize the dynamics of nDNA accumulation, we present two new dynamical/statistical models. We applied these models to the accumulation of the nDNA content of sperm cells in L. barbarum between 16 to 32 hours after pollination in a style. A statistical analysis of experimental data, involving Markov chain Monte Carlo methods, allowed estimation of parameters of the models. We conclude that the model with no variation in the rate of nDNA accumulation adequately summarizes the data. This is the first work where the dynamics of nDNA accumulation has been quantitatively modeled and analyzed.

  13. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer.

    Science.gov (United States)

    Nurwidya, Fariz; Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-09-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer. PMID:27689025

  14. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer

    Science.gov (United States)

    Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-01-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer.

  15. 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)

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Typical Cell Signaling Response to Ionizing Radiation:DNA Damage and Extranuclear Damage

    Institute of Scientific and Technical Information of China (English)

    Hui Yu

    2012-01-01

    To treat many types of cancer,ionizing radiation (IR) is primarily used as external-beam radiotherapy,brachytherapy,and targeted radionuclide therapy.Exposure of tumor cells to IR can induce DNA damage as well as generation of reactiveoxygen species (ROS) and reactive nitrogen species (RNS) which can cause non-DNA lesions or extracellular damage like lipid perioxidation.The initial radiation-induced cell responses to DNA damage and ROS like the proteolytic processing,as well as synthesis and releasing ligands (such as growth factors,cytokines,and hormone) can cause the delayed secondary responses in irradiated and unirradiated bystander cells through paracrine and autocrine pathways.

  2. 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.

  3. Loss of retrovirus production in JB/RH melanoma cells transfected with H-2Kb and TAP-1 genes.

    Science.gov (United States)

    Li, M; Xu, F; Muller, J; Huang, X; Hearing, V J; Gorelik, E

    1999-01-20

    JB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kb molecules due to down-regulation of the H-2Kb and TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kb and cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kb molecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviral PstI restriction site and the loss of a HindIII restriction site in the pol region. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA.

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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.

  9. 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...

  10. 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

  11. 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.

  12. 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.

  13. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    Science.gov (United States)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  14. 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.

  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. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    Energy Technology Data Exchange (ETDEWEB)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Complex forms of mitochondrial DNA in human B cells transformed by Epstein-Barr virus (EBV)

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Christiansen, C; Zeuthen, J

    1983-01-01

    Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed...

  7. 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

  8. Molecular behavior of DNA in a cell-sized compartment coated by lipids

    Science.gov (United States)

    Hamada, Tsutomu; Fujimoto, Rie; Shimobayashi, Shunsuke F.; Ichikawa, Masatoshi; Takagi, Masahiro

    2015-06-01

    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 molecular systems in a small space. These findings may contribute to a better understanding of the physical mechanism of molecular events and reactions inside a cell.

  9. Sequence-selective DNA binding with cell-permeable oligoguanidinium-peptide conjugates.

    Science.gov (United States)

    Mosquera, Jesús; Sánchez, Mateo I; Valero, Julián; de Mendoza, Javier; Vázquez, M Eugenio; Mascareñas, José L

    2015-03-21

    Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugate with cell internalization properties.

  10. Single-tube linear DNA amplification for genome-wide studies using a few thousand cells

    NARCIS (Netherlands)

    Shankaranarayanan, P.; Mendoza-Parra, M.A.; Gool, van W.; Trindade, L.M.; Gronemeyer, H.

    2012-01-01

    Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled

  11. 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…

  12. 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.)

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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)

  2. Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster.

    Science.gov (United States)

    Woodruff, R C; Thompson, J N; Barker, J S; Huai, H

    1999-01-01

    Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  8. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation.

    Science.gov (United States)

    Lowes, Lori E; Bratman, Scott V; Dittamore, Ryan; Done, Susan; Kelley, Shana O; Mai, Sabine; Morin, Ryan D; Wyatt, Alexander W; Allan, Alison L

    2016-09-08

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes.

  9. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation.

    Science.gov (United States)

    Lowes, Lori E; Bratman, Scott V; Dittamore, Ryan; Done, Susan; Kelley, Shana O; Mai, Sabine; Morin, Ryan D; Wyatt, Alexander W; Allan, Alison L

    2016-01-01

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes. PMID:27618023

  10. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation

    Science.gov (United States)

    Lowes, Lori E.; Bratman, Scott V.; Dittamore, Ryan; Done, Susan; Kelley, Shana O.; Mai, Sabine; Morin, Ryan D.; Wyatt, Alexander W.; Allan, Alison L.

    2016-01-01

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes. PMID:27618023

  11. 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.

  12. 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

  13. 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

  14. 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...

  15. 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...

  16. 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.

  17. 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.)

  18. 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 ...

  19. 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...

  20. 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)

  1. 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

  2. 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

  3. 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...

  4. 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...

  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. DNA damage enhanced by the attenuation of SLD5 delays cell cycle restoration in normal cells but not in cancer cells.

    Directory of Open Access Journals (Sweden)

    Zhi-Yuan Gong

    Full Text Available SLD5 is a member of the GINS complex composed of PSF1, PSF2, PSF3 and SLD5, playing a critical role in the formation of the DNA replication fork with CDC45 in yeast. Previously, we had isolated a PSF1 orthologue from a murine hematopoietic stem cell DNA library and were then able to identify orthologues of all the other GINS members by the yeast two hybrid approach using PSF1 as the bait. These GINS orthologues may also function in DNA replication in mammalian cells because they form tetrameric complexes as observed in yeast, and gene deletion mutants of both PSF1 and SLD5 result in a lack of epiblast proliferation and early embryonic lethality. However, we found that PSF1 is also involved in chromosomal segregation in M phase, consistent with recent suggestions that homologues of genes associated with DNA replication in lower organisms also regulate cellular events other than DNA replication in mammalian cells. Here we analyzed the function of SLD5 other than DNA replication and found that it is active in DNA damage and repair. Attenuation of SLD5 expression results in marked DNA damage in both normal cells and cancer cells, suggesting that it protects against DNA damage. Attenuation of SLD5 delays the DNA repair response and cell cycle restoration in normal cells but not in cancer cells. These findings suggest that SLD5 might represent a therapeutic target molecule acting at the level of tumor stromal cells rather than the cancerous cells themselves, because development of the tumor microenvironment could be delayed or disrupted by the suppression of its expression in the normal cell types within the tumor.

  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. Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging.

    Science.gov (United States)

    Chatre, Laurent; Ricchetti, Miria

    2013-02-15

    Mitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial transcription and replication imaging protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: (i) replication initiation active and transcript-positive (Ia-Tp); (ii) replication initiation silent and transcript-positive (Is-Tp); and (iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations that provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.

  9. Biomarkers of radiation or oxidative damage to DNA in cells

    International Nuclear Information System (INIS)

    Major efforts have been devoted during the last two decades to the development of chemical and biochemical assays aimed at monitoring oxidized bases within DNA. Until recently, the level of oxidized bases in cellular DNA was overestimated by factors varying from one to three orders of magnitude. The reasons of these inconsistencies are now identified. Thus, artifactual oxidation of nucleobases may occur during the silylation reaction prior to gas chromatography mass spectrometry analysis and to a lesser extent during DNA extraction. The use of the so-called chaotropic DNA extraction method together with chelating agents was found to significantly prevent spurious DNA oxidation to occur. HPLC separations coupled to either electrochemical detection or versatile electrospray ionization tandem-mass spectrometry appear to be appropriate analytical tools when at least 20 μg of DNA is available. Interestingly, up to 11 modified nucleosides and nucleobases including the four cis and trans diastereomers of 5,6- dihydroxy-5,6-dihydrothymidine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-hydroxy-2'-deoxyuridine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine together with related formamidopyrimidine derivatives were measured in the DNA of neoplastic human monocytes exposed to ionizing radiation using the latter assay. The association of base excision DNA repair enzymes including bacterial formamidopyrimidine glycosylase (Fpg) and endonuclease III (endo III) with the comet assay represents a better alternative for assessing low levels of base damage. The basal level of Fpg- and endo III-sensitive sites was found to be similar, close to 2.1 per 107 bases, within the DNA of human monocytes. The yields of the different classes of damage per Gy and 107 bases are the following: 0.48 Fpg-sensitive sites, 0.53 endo III-sensitive sites and 1.30 strand breaks (direct nicks and alkali-labile sites). Interestingly, the above three classes

  10. 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.

  11. An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells.

    Science.gov (United States)

    Dumas, F; Duckely, M; Pelczar, P; Van Gelder, P; Hohn, B

    2001-01-16

    Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems. PMID:11149937

  12. Light chain editors of anti-DNA receptors in human B cells.

    Science.gov (United States)

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André; Weigert, Martin

    2014-02-10

    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 mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.

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

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    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.

  14. 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

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

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    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.

  16. 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)

  17. 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...

  18. 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.

  19. THAP5 is a DNA binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Science.gov (United States)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla; Goto, Yamafumi; Takata, Minoru; Turkson, James; Li, Xiaoman Shawn; Zervos, Antonis S.

    2011-01-01

    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. PMID:21110952

  20. 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

  1. Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yan Nan; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun-Ran; Kim, Su-Hyeon; Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Park, Sang Jun; Kim, Chun-Ho [Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Lee, Kee-Ho, E-mail: khlee@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2014-01-17

    Highlights: •HAS2 may be a promising target for the radiosensitization of human cancer. •HAS2 is elevated (up to ∼10-fold) in irradiated radioresistant and -sensitive cancer cells. •HAS2 knockdown sensitizes cancer cells to radiation. •HAS2 knockdown potentiates irradiation-induced DNA damage and apoptotic death. •Thus, the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. -- Abstract: Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.

  2. The effect of caffeine and adenine on radiation induced suppression of DNA synthesis, and cell survival

    International Nuclear Information System (INIS)

    Exposure of cultured mammalian cells to ionizing radiation or UV light results in a transient decrease in the rate of DNA synthesis. This depression in synthetic rate may be attenuated or deferred via a post-irradiation treatment with caffeine or adenine. It has been suggested that this attenuation may increase the fixation of damage and, therefore, increase radiation sensitivity. However, it has been previously reported that, for V79 cells treated with caffeine or adenine, no correlation exists between the extent of depression and cell survival. The present investigation expands upon these findings by examining the effect of caffeine or adenine post-irradiation treatment on two cell lines with normal UV sensitivity, mouse 3T3 and CHO AA8 cells, and one UV sensitive cell line, CHO UV5 cells. Both caffeine and adenine have been found to reduce, or delay, the suppression in DNA synthesis in all three cell lines. Surprisingly, caffeine appeared to induced even the UV5 cells to recover DNA synthetic ability. The amount of reduction in suppression of DNA synthesis, however, varies between the different cell lines and no consistent relationship with cell survival has emerged

  3. CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

    International Nuclear Information System (INIS)

    Background: Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. Materials and methods: A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes. Results: A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. Conclusions: CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs

  4. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fung, N.

    1998-03-27

    Since the first demonstration of the laser in the 1960`s, lasers have found numerous applications in analytical chemistry. In this work, two different applications are described, namely, DNA sequencing with capillary gel electrophoresis and single cell analysis with mass spectrometry. Two projects are described in which high-speed DNA separations with capillary gel electrophoresis were demonstrated. In the third project, flow cytometry and mass spectrometry were coupled via a laser vaporization/ionization interface and individual mammalian cells were analyzed. First, DNA Sanger fragments were separated by capillary gel electrophoresis. A separation speed of 20 basepairs per minute was demonstrated with a mixed poly(ethylene oxide) (PEO) sieving solution. In addition, a new capillary wall treatment protocol was developed in which bare (or uncoated) capillaries can be used in DNA sequencing. Second, a temperature programming scheme was used to separate DNA Sanger fragments. Third, flow cytometry and mass spectrometry were coupled with a laser vaporization/ionization interface.

  5. Detection of DNA strand breaks in mammalian cells using the radioresistant bacterium PprA protein

    International Nuclear Information System (INIS)

    We have previously found that the PprA protein from Deinococcus radiodurans possesses ability to recognize DNA carrying strand breaks. In the present study, we attempted to visualize radiation-induced DNA strand breaks with PprA protein using immunofluorescence technique to elucidate the DNA damage response mechanism in mammalian cultured cells. As a result, colocalization of Cy2 and DAPI fluorescent signals was observed. This observation suggests that DNA strand breaks in the nucleus of CHO-K1 cells were effectively detected using the PprA protein. The amount of DNA strand breaks (integrated density of Cy2 fluorescent signals) was increased with the increase in the radiation dose. (author)

  6. Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC

    DEFF Research Database (Denmark)

    Dowler Nygaard, Anneli; Spindler, Karen-Lise Garm; Pallisgaard, Niels;

    2014-01-01

    Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can be...... analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty‑nine patients...... were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma...

  7. Helicobacter pylori infection affects mitochondrial function and DNA repair, thus, mediating genetic instability in gastric cells

    DEFF Research Database (Denmark)

    Machado, Ana Manuel Dantas; Madsen, Claus Desler; Bøggild, Cecilie Sisse Line;

    2013-01-01

    Helicobacter pylori infection is an important factor for the development of atrophic gastritis and gastric carcinogenesis. However, the mechanisms explaining the effects of H. pylori infection are not fully elucidated. H. pylori infection is known to induce genetic instability in both nuclear...... and mitochondrial DNA of gastric epithelial cells. The mutagenic effect of H. pylori infection on nuclear DNA is known to be a consequence, in part, of a down-regulation of expression and activity of major DNA repair pathways. In this study, we demonstrate that H. pylori infection of gastric adenocarcinoma cells....... pylori infection, furthermore, the results demonstrate that multiple DNA repair activities are involved in protecting mtDNA during infection....

  8. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma

    Science.gov (United States)

    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 chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management. PMID:26918050

  9. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma.

    Science.gov (United States)

    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 chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management. PMID:26918050

  10. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

    International Nuclear Information System (INIS)

    The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v1, a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

  11. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    Science.gov (United States)

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  12. Hiwi Promotes the Proliferation of Colorectal Cancer Cells via Upregulating Global DNA Methylation

    Directory of Open Access Journals (Sweden)

    Lin Yang

    2015-01-01

    Full Text Available Hiwi is well known for its role in stem cell renewal, maintaining the resting stage, and downregulating cell cycle of stem cells via RNA silencing. And Hiwi overexpression has been recognized in several types of cancers. In the present study, we examined the Hiwi expression in colorectal cancer (CRC specimens in both mRNA and protein levels via real-time quantitative PCR, western blot assay, and immunohistochemical staining. Then we explored the role of Hiwi in the cancer cell proliferation and in the DNA methylation in human CRC Caro-2 and HT-29 cell lines. Results demonstrated that both mRNA and protein levels of Hiwi were significantly higher in 38 CRC tissues than in 38 peritumor tissues. Moreover, the Hiwi overexpression with an adenovirus vector significantly promoted the proliferation of Caro-2 and HT-29 cells, associated with significant increase in the global DNA methylation levels. And the chemical inhibition of DNA methylation significantly restrained such proliferation promotion. In summary, we confirmed that Hiwi was overexpressed in CRC tissues and that the forced Hiwi overexpression promoted the proliferation and global DNA methylation of CRC cell lines. Our results imply for the first time that Hiwi promotes the proliferation of CRC cells via promoting global DNA methylation.

  13. Curcumin alters gene expression-associated DNA damage, cell cycle, cell survival and cell migration and invasion in NCI-H460 human lung cancer cells in vitro.

    Science.gov (United States)

    Chiang, I-Tsang; Wang, Wei-Shu; Liu, Hsin-Chung; Yang, Su-Tso; Tang, Nou-Ying; Chung, Jing-Gung

    2015-10-01

    Lung cancer is the most common cause of cancer mortality and new cases are on the increase worldwide. However, the treatment of lung cancer remains unsatisfactory. Curcumin has been shown to induce cell death in many human cancer cells, including human lung cancer cells. However, the effects of curcumin on genetic mechanisms associated with these actions remain unclear. Curcumin (2 µM) was added to NCI-H460 human lung cancer cells and the cells were incubated for 24 h. Total RNA was extracted from isolated cells for cDNA synthesis, labeling, microarray hybridization and flour‑labeled cDNA hybridized on chip. Localized concentrations of fluorescent molecules were detected and quantified using Expression Console software (Affymetrix) with default RMA parameters. GeneGo software was used for the key genes involved and their possible interaction pathways. The results showed that ~170 genes were significantly upregulated and 577 genes were significantly downregulated in curcumin‑treated cells. Specifically, the up‑ and downregulated genes included CCNE2, associated with DNA damage; ID3, associated with cell survival and 146 genes with a >2- to 3-fold change including the TP53INP1 gene, associated with DNA damage; CDC6, CDCA5, TAKMIP2, CDK14, CDK5, CDCA76, CDC25A, CDC5L and SKP2, associated with cell cycle; the CARD6, ID1 and ID2 genes, associated with cell survival and the BRMS1L, associated with cell migration and invasion. Additionally, 59 downregulated genes exhibited a >4-fold change, including the DDIT3 gene, associated with DNA damage; while 97 genes had a >3- to 4-fold change including the DDIT4 gene, associated with DNA damage; the CCPG1 gene, associated with cell cycle and 321 genes with a >2- to 3-fold including the GADD45A and CGREF1 genes, associated with DNA damage; the CCPG1 gene, associated with cell cycle, the TNFRSF10B, GAS5, TSSC1 and TNFRSF11B gene, associated with cell survival and the ARHAP29 and CADM2 genes, associated with cell migration

  14. Evidence for existence and compactness of DNA superstructure units in mammalian cells: A microdosimetric approach to radiation - induced DNA release assayed by neutral sucrose gradient sedimentation

    International Nuclear Information System (INIS)

    Experimental results obtained with the neutral sucrose gradient sedimentation techniques of analysing mammalian DNA after irradiation in vivo (Chinese hamster cells V79) are evaluated theoretically in order to verify and extend a model by Ormerod and Lehmann that describes the gradual release of free DNA from the DNA-membrane complex. The model is based on the idea of chromatin organization in the form of membrane attached superstructure unit (MASSUs) defined by consecutive attachment points in intervals of M0 (DNA molecular weight of a MASSU). DNA sedimentation after cell lysis with sarkosyl as detergent allows good separation of the released free DNA from that remaining in the complex. The dose-dependence of both the percentage of DNA released and pertinent molecular weight parameters as measured with X-rays and derived from the model confirms it and yields the MASSU size M0=2.4x109 g/mol. (orig./WL)

  15. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

  16. Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

    OpenAIRE

    Liang, T J; Makdisi, W J; Sun, S.; Hasegawa, K.; Zhang, Y.; Wands, J R; Wu, C. H.; Wu, G Y

    1993-01-01

    A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomuc...

  17. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

    Directory of Open Access Journals (Sweden)

    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  18. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Edith E., E-mail: ed.mueller@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Mayr, Johannes A., E-mail: h.mayr@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Zimmermann, Franz A., E-mail: f.zimmermann@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Feichtinger, Rene G., E-mail: r.feichtinger@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Stanger, Olaf, E-mail: o.stanger@rbht.nhs.uk [Department of Cardiac Surgery, Paracelsus Medical University, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Sperl, Wolfgang, E-mail: w.sperl@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Kofler, Barbara, E-mail: b.kofler@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  19. Induction and repair of DNA strand breaks in human tumor cells

    International Nuclear Information System (INIS)

    The presence of radioresistant or repair-proficient cells in a tumor is often associated with radiotherapy failure. The authors have begun to study the mechanisms of resistance in these tumor cells. Their initial studies focused on the induction and repair of DNA strand breaks as measured by DNA elution. Eight human tumor (5 squamous cell carcinomas, 2 melanomas, and 1 adenocarcinoma) and 2 nonmalignant cell lines have been examined. Their D/sub O/s range from 1.07 Gy to 2.81 Gy while their extrapolation numbers (n) range from 1.2 to 24.8. Three parameters are being investigated for X-ray-induced DNA single and double-strand breaks; 1) the initial number of breaks induced, 2) the residual DNA strand break frequency and 3) the time to repair 50% of the lesions. The kinetics of repair of DNA single-strand breaks were similar for all the lines studied. In 2 cases, radiosensitivity was associated with either a high residual DNA strand break frequency or a slower rate of repair. No single parameter or combination of parameters, however, consistently correlated with radiosensitivity or n. Thus, while differences in the induction and repair of DNA single-strand breaks might explain some of the observed differences in radiation responses, they are in general a poor predictor radiation sensitivity

  20. Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

    Science.gov (United States)

    Liang, T J; Makdisi, W J; Sun, S; Hasegawa, K; Zhang, Y; Wands, J R; Wu, C H; Wu, G Y

    1993-01-01

    A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions. Images PMID:8383700

  1. RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA FROM HUMAN LUNG ADENOCARCINOMA CELL LINE SPC-A-1

    Institute of Scientific and Technical Information of China (English)

    HU Yide; QIAN Guisheng; CHEN Weizhong; LI Shuping; WANG Guansong; MAO Baoling

    1999-01-01

    Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease, which were Pvu Ⅱ, Xho Ⅰ, Pst Ⅰ, EcoR Ⅰ,BstE Ⅱ, Hind Ⅲ, Hpa Ⅰ, Bcl Ⅰ, EcoR Ⅴ, Sca Ⅰ and Xba Ⅰ.Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double-digestion method.Results: It was found that no variation at 32 restrictionsites could be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276 (EcoR Ⅴ) within the noncoding region.Conclusion: These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly stable. While the major variation of nucleotide is probably located in the noncoding region.

  2. An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ermakov, Aleksei V., E-mail: avePlato@mail.ru [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Konkova, Marina S.; Kostyuk, Svetlana V.; Smirnova, Tatiana D.; Malinovskaya, Elena M.; Efremova, Liudmila V.; Veiko, Natalya N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2011-07-01

    The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10 cGy. The fragments of extracellular genomic DNA (ecDNA{sup R}) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA{sup R}, followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA{sup R} disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA{sup R}) through the culture medium.

  3. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  4. Age-related mitochondrial DNA depletion and the impact on pancreatic Beta cell function.

    Directory of Open Access Journals (Sweden)

    Donna L Nile

    Full Text Available Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes.

  5. File list: Oth.NoD.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.10.DNA-RNA_hybrids.AllCell.bed ...

  6. File list: Oth.NoD.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.50.DNA-RNA_hybrids.AllCell.bed ...

  7. File list: Oth.NoD.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.05.DNA-RNA_hybrids.AllCell.bed ...

  8. File list: Oth.NoD.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.20.DNA-RNA_hybrids.AllCell.bed ...

  9. Plasma cell-free DNA levels are elevated in acute Puumala hantavirus infection.

    Directory of Open Access Journals (Sweden)

    Tuula K Outinen

    Full Text Available INTRODUCTION: Puumala hantavirus (PUUV causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE. The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease. METHODS: Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine. RESULTS: The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94-3.29 µg/ml vs. median 0.77 µg/ml, range 0.55-0.99 µg/ml, P<0.001. The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count (r = 0.388, P = 0.002 and the length of hospital stay (r = 0.376, P = 0.003, and inversely with minimum blood platelet count (r = -0.297, P = 0.020. Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150-200 bp. The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA (r = 0.513, P = 0.021. Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level. CONCLUSIONS: The plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.

  10. Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model

    Institute of Scientific and Technical Information of China (English)

    Lu Yang; Huijun Wang; Xin Luo; Pengliang Mao; Weidong Tian; Yujiang Shi; Guoying Huang; Jin Zhang; Duan Ma

    2012-01-01

    DNA methylation and demethylation play important roles in mediating epigenetic regulation.So far,the mechanism of DNA demethylation remains elusive and controversial.Here,we constructed a plasmid,named with pCBS-luc,that contained an artificial CpG island,eight Gal4 DNA-binding domain binding site,an SV40 promoter,and a firefly luciferase reporter gene.The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells.The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained.The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification.The methylation status could be maintained even after 15 passages.The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs.Using our me-pCBS-luc model,we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells.Altogether,we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid,and in this model we found that VP16 could lead to DNA demethylation.We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.

  11. Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells

    Directory of Open Access Journals (Sweden)

    Schins Roel PF

    2009-06-01

    Full Text Available Abstract Titanium dioxide (TiO2, also known as titanium (IV oxide or anatase, is the naturally occurring oxide of titanium. It is also one of the most commercially used form. To date, no parameter has been set for the average ambient air concentration of TiO2 nanoparticles (NP by any regulatory agency. Previously conducted studies had established these nanoparticles to be mainly non-cyto- and -genotoxic, although they had been found to generate free radicals both acellularly (specially through photocatalytic activity and intracellularly. The present study determines the role of TiO2-NP (anatase, ∅ in vitro. For comparison, iron containing nanoparticles (hematite, Fe2O3, ∅ 2-NP did not induce DNA-breakage measured by the Comet-assay in both cell types. Generation of reactive oxygen species (ROS was measured acellularly (without any photocatalytic activity as well as intracellularly for both types of particles, however, the iron-containing NP needed special reducing conditions before pronounced radical generation. A high level of DNA adduct formation (8-OHdG was observed in IMR-90 cells exposed to TiO2-NP, but not in cells exposed to hematite NP. Our study demonstrates different modes of action for TiO2- and Fe2O3-NP. Whereas TiO2-NP were able to generate elevated amounts of free radicals, which induced indirect genotoxicity mainly by DNA-adduct formation, Fe2O3-NP were clastogenic (induction of DNA-breakage and required reducing conditions for radical formation.

  12. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

    Energy Technology Data Exchange (ETDEWEB)

    Pellicciari, C.; Mangiarotti, R.; Bottone, M.G.; Danova, M. [Univ. of Pavia (Italy); Wang, E. [Jewish General Hospital, Montreal, Quebec (Canada)

    1995-12-01

    Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.

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

    Directory of Open Access Journals (Sweden)

    Mahmoud Aljurf

    2016-01-01

    Full Text Available 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 leukemia patients (N = 126 showed that, of 84 patients with 100% donor DNA in PMN, 16 (19% had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19% showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41% showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse.

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

    Science.gov (United States)

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

    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 leukemia patients (N = 126) showed that, of 84 patients with 100% donor DNA in PMN, 16 (19%) had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse. PMID:27006832

  15. Relationship between transformation of human embryo lung cell and DNA strand break induced by γ-irradiation

    International Nuclear Information System (INIS)

    γ-irradiation effects on transformation and DNA strand break of human embryo lung cell were studied by means of cell and molecular biology. The cell transformation appeared at 20 th passage post 0.25∼5.0 Gy γ-irradiation. The transformation was relative to doses and post-time of irradiation. DNA strand break was detected by NTA (nick translation assay). The result indicated DNA strand break was arisen from γ ray with different doses. DNA strand break also was relative to radiation doses as cell transformation. Correlation between DNA strand break and cell transformation was analysed. The analysis indicated that effect of cell transformation enhanced with increase of DNA strand break. It was suggested that carcinogenesis by radiation is closely relative to DNA damage

  16. Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    The repair of DNA damage produced by 137Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution. The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G. Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation. At later repair times, 12-17 h, more ESS remained in XP than in normal cells. The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced. The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive

  17. Cytotoxicity and DNA crosslinks produced by mitomycin analogs in aerobic and hypoxic EMT6 cells.

    Science.gov (United States)

    Keyes, S R; Loomis, R; DiGiovanna, M P; Pritsos, C A; Rockwell, S; Sartorelli, A C

    1991-01-01

    Several mitomycin antibiotics were evaluated for their capacities to kill EMT6 tumor cells and to produce DNA crosslinks under conditions of oxygenation and hypoxia. The agents examined included mitomycin C, porfiromycin, and the 7-aminomethyl dithioacetal derivative of mitomycin C (BMY-43324), all of which caused greater kill of hypoxic cells than of their oxygenated counterparts; the N,N'-dimethylaminomethylene derivative of mitomycin C (BMY-25282), which was considerably more cytotoxic under oxygenated conditions than in hypoxia; and the N,N'-dimethylaminomethylene derivative of porfiromycin (BL-6783), which was equal in its toxicity to hypoxic and oxygenated cells. All of these agents produced DNA crosslinks in EMT6 cells, as measured by alkaline elution. The number of crosslinks required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation, suggesting that the crosslinking of DNA was a major lesion in the cytodestructive action of the mitomycins. PMID:1760250

  18. Artesunate Reduces Proliferation, Interferes DNA Replication and Cell Cycle and Enhances Apoptosis in Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded.The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated.DNA replication was evaluated by [3 H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.

  19. Effect of different gravity environments on DNA fragmentation and cell death in Kalanchoe leaves.

    Science.gov (United States)

    Pedroso, M C; Durzan, D J

    2000-11-01

    Different gravity environments have been shown to significantly affect leaf-plantlet formation and asexual reproduction in Kalanchoë daigremontiana Ham. and Perr. In the present work, we investigated the effect of gravity at tissue and cell levels. Leaves and leaf-plantlets were cultured for different periods of time (min to 15 d) in different levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 x 10(-4) g), 1 g (control) and hypergravity (centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity affected cell death (apoptosis) in this species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO) formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis, mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal differentiation. Exposures to acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with N(G)-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how chloroplasts, cells and leaves may respond (and adapt) to gravity changes. PMID:11762440

  20. 45. Damage effects of sulfur dioxide inhalation on DNA of brain cells from mice

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The damage effects of sulfur dioxide (SO2) inhalation on DNA of brain cells from mice were studied with the single cell microgel electrophoresis tecknique (Comet test). The results show that SO2 inhalation caused the damage effects to DNA of the mouse brain cells in a dose-dependent manner. The results indicate that even under SO2 inhalation at low concentrations as 7 mg SO2/m3, The brain cells with DNA damaged also reached to 98.8%, it implies the brain cells of mammalian animals are very sensitive to SO2 inhalation. The results also indicate that DNA damage of the brain cells from male mice is more serious than that from female mice, that remains to be further studied. These results led us to conclusion SO2 pollution even at low concentrations also has a potential risk to damage genetic material DNA of brain cells from mammalian animals. It might be explained by our conclusion that the recently published epidemiological studies of workers exposed to SO2 or it's derivatives (bi)sulfite) found increased mortality for brain cancer.

  1. Monitoring of organ transplants through genomic analyses of circulating cell-free DNA

    Science.gov (United States)

    de Vlaminck, Iwijn

    Solid-organ transplantation is the preferred treatment for patients with end-stage organ diseases, but complications due to infection and acute rejection undermine its long-term benefits. While clinicians strive to carefully monitor transplant patients, diagnostic options are currently limited. My colleagues and I in the lab of Stephen Quake have found that a combination of next-generation sequencing with a phenomenon called circulating cell-free DNA enables non-invasive diagnosis of both infection and rejection in transplantation. A substantial amount of small fragments of cell-free DNA circulate in blood that are the debris of dead cells. We discovered that donor specific DNA is released in circulation during injury to the transplant organ and we show that the proportion of donor DNA in plasma is predictive of acute rejection in heart and lung transplantation. We profiled viral and bacterial DNA sequences in plasma of transplant patients and discovered that the relative representation of different viruses and bacteria is informative of immunosuppression. This discovery suggested a novel biological measure of a person's immune strength, a finding that we have more recently confirmed via B-cell repertoire sequencing. Lastly, our studies highlight applications of shotgun sequencing of cell-free DNA in the broad, hypothesis free diagnosis of infection.

  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

    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...... maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells....

  3. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  4. DNA apoptosis and stability in B-cell chronic lymphoid leukaemia: implication of the DNA double-strand breaks repair system by non homologous recombination

    International Nuclear Information System (INIS)

    After an introduction presenting the diagnosis and treatment of chronic lymphoid leukaemia, its molecular and genetic characteristics, and its cellular origin and clonal evolution, this research thesis describes the apoptosis (definition and characteristics, cancer and chemotherapy, apoptotic ways induced by gamma irradiation), the genotoxic stresses, the different repair mechanisms for different damages, and the DNA repair processes. It reports how human chronic lymphocytic leukaemia B cells can escape DNA damage-induced apoptosis through the non-homologous end-joining DNA repair pathway, and presents non-homologous end-joining DNA repair as a potent mutagenic process in human chronic lymphocytic leukaemia B cells

  5. DNA Methylation and Apoptosis Resistance in Cancer Cells

    OpenAIRE

    Pierre-François Cartron; François Marie Vallette; Eric Hervouet; Mathilde Cheray

    2013-01-01

    Apoptosis is a cell death programme primordial to cellular homeostasis efficiency. This normal cell suicide program is the result of the activation of a cascade of events in response to death stimuli. Apoptosis occurs in normal cells to maintain a balance between cell proliferation and cell death. A deregulation of this balance due to modifications in the apoptosic pathway leads to different human diseases including cancers. Apoptosis resistance is one of the most important hallmarks of cance...

  6. DNA methylation in stem cell renewal and multipotency

    OpenAIRE

    Berdasco, María; Esteller, Manel

    2011-01-01

    Owing to their potential for differentiation into multiple cell types, multipotent stem cells extracted from many adult tissues are an attractive stem cell resource for the replacement of damaged tissues in regenerative medicine. The requirements for cellular differentiation of an adult stem cell are a loss of proliferation potential and a gain of cell-type identity. These processes could be restricted by epigenetic modifications that prevent the risks of lineage-unrelated gene expression or ...

  7. Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture

    OpenAIRE

    Deora, Ami A.; Diaz, Fernando; Schreiner, Ryan; Rodriguez-Boulan, Enrique

    2007-01-01

    Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to ...

  8. Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

    Science.gov (United States)

    Yao, N; Hurwitz, J; O'Donnell, M

    2000-01-14

    Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed. PMID:10625694

  9. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Seok-Jo Kim

    2015-09-01

    Full Text Available 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 evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1 and mitochondrial aconitase (ACO-2 in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

  10. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis.

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; 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 evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

  11. Identification of Circulating Tumor DNA for the Early Detection of Small-cell Lung Cancer.

    Science.gov (United States)

    Fernandez-Cuesta, Lynnette; Perdomo, Sandra; Avogbe, Patrice H; Leblay, Noemie; Delhomme, Tiffany M; Gaborieau, Valerie; Abedi-Ardekani, Behnoush; Chanudet, Estelle; Olivier, Magali; Zaridze, David; Mukeria, Anush; Vilensky, Marta; Holcatova, Ivana; Polesel, Jerry; Simonato, Lorenzo; Canova, Cristina; Lagiou, Pagona; Brambilla, Christian; Brambilla, Elisabeth; Byrnes, Graham; Scelo, Ghislaine; Le Calvez-Kelm, Florence; Foll, Matthieu; McKay, James D; Brennan, Paul

    2016-08-01

    Circulating tumor DNA (ctDNA) is emerging as a key potential biomarker for post-diagnosis surveillance but it may also play a crucial role in the detection of pre-clinical cancer. Small-cell lung cancer (SCLC) is an excellent candidate for early detection given there are no successful therapeutic options for late-stage disease, and it displays almost universal inactivation of TP53. We assessed the presence of TP53 mutations in the cell-free DNA (cfDNA) extracted from the plasma of 51 SCLC cases and 123 non-cancer controls. We identified mutations using a pipeline specifically designed to accurately detect variants at very low fractions. We detected TP53 mutations in the cfDNA of 49% SCLC patients and 11.4% of non-cancer controls. When stratifying the 51 initial SCLC cases by stage, TP53 mutations were detected in the cfDNA of 35.7% early-stage and 54.1% late-stage SCLC patients. The results in the controls were further replicated in 10.8% of an independent series of 102 non-cancer controls. The detection of TP53 mutations in 11% of the 225 non-cancer controls suggests that somatic mutations in cfDNA among individuals without any cancer diagnosis is a common occurrence, and poses serious challenges for the development of ctDNA screening tests. PMID:27377626

  12. Uptake of Single-Walled Carbon Nanotubes Conjugated with DNA by Microvascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Joseph Harvey

    2012-01-01

    Full Text Available Single-walled carbon nanotubes (SWCNTs have been proposed to have great therapeutic potential. SWCNTs conjugated with drugs or genes travel in the systemic circulation to reach target cells or tissues following extravasation from microvessels although the interaction between SWCNT conjugates and the microvascular endothelial cells (ECs remains unknown. We hypothesized that SWCNT-DNA conjugates would be taken up by microvascular ECs and that this process would be facilitated by SWCNTs compared to facilitation by DNA alone. ECs were treated with various concentrations of SWCNT-DNA-FITC conjugates, and the uptake and intracellular distribution of these conjugates were determined by a confocal microscope imaging system followed by quantitative analysis of fluorescence intensity. The uptake of SWCNT-DNA-FITC conjugates (2 μg/mL by microvascular ECs was significantly greater than that of DNA-FITC (2 μg/mL, observed at 6 hrs after treatment. For the intracellular distribution, SWCNT-DNA-FITC conjugates were detected in the nucleus of ECs, while DNA-FITC was restricted to the cytoplasm. The fluorescence intensity and distribution of SWCNTs were concentration and time independent. The findings demonstrate that SWCNTs facilitate DNA delivery into microvascular ECs, thus suggesting that SWCNTs serving as drug and gene vehicles have therapeutic potential.