WorldWideScience

Sample records for cell proviral dna

  1. A genotypic HIV-1 proviral DNA coreceptor tropism assay: characterization in viremic subjects

    Science.gov (United States)

    2014-01-01

    Background HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test. Methods Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR). Results Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%–98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86). Conclusions Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes. PMID:24904682

  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)

    Janowski, M.; Merregaert, J.; Nuyten, J.M.; Maisin, J.R.

    1984-01-01

    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 [fr

  3. HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

    Science.gov (United States)

    Nascimento-Brito, Sieberth; Paulo Zukurov, Jean; Maricato, Juliana T; Volpini, Angela C; Salim, Anna Christina M; Araújo, Flávio M G; Coimbra, Roney S; Oliveira, Guilherme C; Antoneli, Fernando; Janini, Luiz Mário R

    2015-01-01

    In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

  4. HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

    Directory of Open Access Journals (Sweden)

    Sieberth Nascimento-Brito

    Full Text Available In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5 and C-X-C chemokine Receptor type 4 (CXCR4 Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

  5. Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment

    Science.gov (United States)

    Massanella, Marta; Richman, Douglas D.; Little, Susan J.; Spina, Celsa A.; Vargas, Milenka V.; Lada, Steven M.; Daar, Eric S.; Dube, Michael P.; Haubrich, Richard H.; Morris, Sheldon R.; Smith, Davey M.

    2014-01-01

    ABSTRACT Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in

  6. HIV drug resistance mutations in proviral DNA from a community treatment program.

    Directory of Open Access Journals (Sweden)

    Anne Derache

    Full Text Available Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations.The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu.Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%; the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26% with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively.The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence.

  7. Autoradiographic studies on proviral DNA synthesis in enucleated chick embryo fibroblasts infected with avian sarcoma virus

    International Nuclear Information System (INIS)

    Tanaka, Terukazu

    1977-01-01

    After infection of RNA tumor viruses to susceptible cells, viral RNA is reversely transcribed into proviral DNA. In order to disclose the site of proviral DNA synthesis in the cells, chick embryo fibroblasts (CEF) were enucleated by centrifugation in the presence of cytochalasin B, and the enucleated CEF (cytoplasts) were infected with B77 strain of avian sarcoma virus (B77-ASV). Incorporation of 3 H-thymidine into DNA in the cytoplasts was investigated autoradiography. Photopositive grains were observed in cytoplasts infected with B77-ASV, but not in mock-infected cytoplasts. The photpositive grains in the cytoplasts infected with B77-ASV disappeared almost completely by DNase I treatment. N-demethyl rifampicin, which is a specific inhibitor of reverse transcriptase, inhibited the appearance of photpositive grains. The B77-ASV-infected cytoplasts were ultrathinsectioned for electron microscopic autoradiography. The photopositive grains appeared in the cytoplasm without relation to mitochondria. These results indicate that the proviral DNA synthesis is initiated in the cytoplasm of B77-ASV-infected chick embryo fibroblast without the direct participation of nucleus. (auth.)

  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...

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

    Science.gov (United States)

    Yin, Qian Qian; Li, Zhen Peng; Zhao, Hai; Pan, Dong; Wang, Yan; Xu, Wei Si; Xing, Hui; Feng, Yi; Jiang, Shi Bo; Shao, Yi Ming; Ma, Li Ying

    2016-04-01

    To 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. Forty-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. Compared 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, PDNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Compared with viral RNA, the distinctive information of DRMs and 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. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  10. Development of a nested PCR assay to detect equine infectious anemia proviral DNA from peripheral blood of naturally infected horses.

    Science.gov (United States)

    Dong, Jian-Bao; Zhu, Wei; Cook, Frank R; Goto, Yoshitaka; Horii, Yoichiro; Haga, Takeshi

    2012-11-01

    Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed based on conserved 5' regions of the viral genome extending from the LTR to the tat gene. Preliminary studies demonstrated that the method has a detection limit of 10 genomic copies and, when applied to a naturally EIAV-infected feral horse population, shows 100 % correlation with conventional serological diagnostic techniques. This assay provides a powerful new tool in the control of EIAV.

  11. Comparison of HTLV-I Proviral Load in Adult T Cell Leukemia/Lymphoma (ATL), HTLV-I-Associated Myelopathy (HAM-TSP) and Healthy Carriers.

    Science.gov (United States)

    Akbarin, Mohammad Mehdi; Rahimi, Hossein; Hassannia, Tahereh; Shoja Razavi, Ghazaleh; Sabet, Faezeh; Shirdel, Abbas

    2013-03-01

    Human T Lymphocyte Virus Type one (HTLV-I) is a retrovirus that infects about 10-20 million people worldwide. Khorasan province in Iran is an endemic area. The majority of HTLV-I-infected individuals sustain healthy carriers but small proportion of infected population developed two progressive diseases: HAM/TSP and ATL. The proviral load could be a virological marker for disease monitoring, therefore in the present study HTLV-I proviral load has been evaluated in ATL and compared to HAM/TSP and healthy carriers. In this case series study, 47 HTLV-I infected individuals including 13 ATL, 23 HAM/TSP and 11 asymptomatic subjects were studied. Peripheral blood mononuclear cells (PBMCs) were investigated for presence of HTLV-I DNA provirus by PCR using LTR and Tax fragments. Then in infected subjects, HTLV-I proviral load was measured using real time PCR TaqMan method. The average age of patients in ATL was 52±8, in HAM/TSP 45.52±15.17 and in carrier's 38.65±14.9 years which differences were not statistically significant. The analysis of data showed a significant difference in mean WBC among study groups (ATL vs HAM/TSP and carriers P=0.0001). Moreover, mean HTLV-I proviral load was 11967.2 ± 5078, 409 ± 71.3 and 373.6 ± 143.3 in ATL, HAM/TSP and Healthy Carriers, respectively. The highest HTLV-I proviral load was measured in ATL group that had a significant correlation with WBC count (R=0.495, P=0.001). The proviral load variations between study groups was strongly significant (ATL vs carrier P=0.0001; ATL vs HAM/TSP P= 0.0001 and HAM/TSP vs carriers Pregime.

  12. Possible involvement of distinct phylogenetic clusters of HIV-1 variants in the discrepancies between coreceptor tropism predictions based on viral RNA and proviral DNA.

    Science.gov (United States)

    Kotani, Hiroshi; Sudo, Koji; Hasegawa, Naoki; Fujiwara, Hiroshi; Hayakawa, Tomohisa; Iketani, Osamu; Yamaguchi, Masaya; Mochizuki, Mayumi; Iwata, Satoshi; Kato, Shingo

    2016-01-01

    The coreceptor tropism testing should be conducted prior to commencing a regimen containing a CCR5 antagonist for treatment of HIV-1 infection. For aviremic patients on long antiretroviral therapy, proviral DNA is often used instead of viral RNA in genotypic tropism testing. However, the tropism predictions from RNA and DNA are sometimes different. We examined the cause of the discrepancies between HIV-1 tropism predictions based on viral RNA and proviral DNA. The nucleotide sequence of the env C2V3C3 region was determined using pair samples of plasma RNA and peripheral blood mononuclear cell DNA from 50 HIV-1 subtype B-infected individuals using population-based sequencing. The samples with discrepant tropism assessments between RNA and DNA were further analyzed using deep sequencing, followed by phylogenetic analysis. The tropism was assessed using the algorithm geno2pheno with a false-positive rate cutoff of 10 %. In population-based sequencing, five of 50 subjects showed discrepant tropism predictions between their RNA and DNA samples: four exhibited R5 tropism in RNA and X4 tropism in DNA, while one exhibited the opposite pattern. In the deep sequencing and phylogenetic analysis, three subjects had single clusters comprising of RNA- and DNA-derived sequences that were a mixture of R5 and X4 sequences. The other two subjects had two and three distinct phylogenetic clusters of sequences, respectively, each of which was dominated by R5 or X4 sequences; sequences of the R5-dominated cluster were mostly found in RNA, while sequences of the X4-dominated cluster were mostly in DNA. Some of HIV-1 tropism discrepancies between viral RNA and proviral DNA seem to be caused by phylogenetically distinct clusters which resides in plasma and cells in different frequencies. Our findings suggest that the tropism testing using PBMC DNA or deep sequencing may be required when the viral load is not suppressed or rebounds in the course of a CCR5 antagonist-containing regimen.

  13. Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

    Science.gov (United States)

    Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

    2018-04-11

    The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Rodrigo Pessôa

    Full Text Available An improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM in therapy-naïve HIV-1-infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs.The MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS or "detuned" enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2 and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses.No TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR and reverse transcriptase (RT regions of the HIV-1 pol gene was 44.5% (57/128, including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI. Of the 57 subjects, 43 (75.4% harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7% had detectable minority resistant variants to NRTIs, 6 (13.9% to NNRTIs, and 16 (37.2% to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis.Our findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these

  15. Evolution of Specific Antibodies and Proviral DNA in Milk of Small Ruminants Infected by Small Ruminant Lentivirus

    Directory of Open Access Journals (Sweden)

    Ana Domenech

    2013-10-01

    Full Text Available The diagnosis of Small Ruminant Lentivirus (SRLV is based on clinical signs, pathological lesions and laboratory testing. No standard reference test for the diagnosis of maedi visna has been validated up to the present, and it is puzzling that tests which detect antibodies against the virus and tests which detect the proviral genome may render opposite results. The aim of this study was to evaluate the presence in milk throughout a lactation period of specific antibodies by ELISA and of SRLV proviral DNA by a PCR of the highly conserved pol region. A six-month study was conducted with the milk of 28 ewes and 31 goats intensively reared. The percentage of animals with antibodies against SRLV increased throughout the study period. Seroprevalence in sheep was 28% at the beginning of the study and by the end it had increased up to 52.4%. In goats, initial seroprevalence of 5.6% increased to 16%. The percentage of PCR positive ewes was stable throughout the study period. Of the positive sheep, 21.4% were PCR-positive before antibodies could be detected and most of them became PCR-negative shortly after the first detection of antibodies. This might suggest that antibodies have a neutralizing effect. In addition, an equal percentage of sheep were always PCR-negative but either became ELISA-positive or was always ELISA-positive, which might support this hypothesis. On the other hand, the PCR results in goats did not follow any pattern and oscillated between 35.3% and 55.6% depending on the month. Most goats positive by PCR failed to develop antibodies in the 6 months tested. We may conclude that the infection and the antibody response to it follow a different trend in sheep and goats.

  16. Genotypic Tropism Testing in HIV-1 Proviral DNA Can Provide Useful Information at Low-Level Viremia

    Science.gov (United States)

    Fabeni, Lavinia; Berno, Giulia; Svicher, Valentina; Ceccherini-Silberstein, Francesca; Gori, Caterina; Bertoli, Ada; Mussini, Cristina; Lichtner, Miriam; Zaccarelli, Mauro; Ammassari, Adriana; Pinnetti, Carmela; Cicalini, Stefania; Mastroianni, Claudio Maria; Andreoni, Massimo; Antinori, Andrea; Perno, Carlo Federico

    2015-01-01

    The possibility of performing genotypic tropism testing (GTT) with proviral DNA (pvDNA) even during suppressed viremia would facilitate the use of CCR5 inhibitors as part of switching, simplification, or intensification strategies. Thus, we aimed to evaluate the tropism concordance between plasma RNA and pvDNA samples and to assess which factors could affect possible discrepancies between the two compartments. GTT was performed using both plasma RNA and pvDNA from 55 sample pairs from drug-experienced patients. Potential differences between the two compartments were evaluated by analyzing coreceptor usage and genetic variability. Paired samples were also stratified in three levels of viremia (500 copies/ml). Overall, Geno2Pheno comparisons of false-positive rates in the two compartments showed good correlation (r = 0.72). A high level of concordance in tropism predictions for the two compartments was found (46/55 sample pairs [83.6%]). Among the 9 sample pairs with discordant tropisms, a larger proportion of pvDNA samples harboring CXCR4/dual-mixed-tropic viruses was found, in comparison with plasma RNA samples (88.9% versus 11.1%; P = 0.0034). Discordant samples were characterized by greater genetic variability than were concordant samples. With stratification of the paired samples according to viremia levels, the prevalence of discordant samples decreased with increasing viremia (500 copies/ml, 6.7%; P = 0.2). Our findings confirm that prediction of viral tropism using pvDNA is feasible even in low-level viremia and provides useful information for therapy optimization for patients with low or suppressed viremia. PMID:26135872

  17. 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, M R; 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...

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

    Science.gov (United States)

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

    2013-01-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism laboratories before embarking on clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/NCT01384682?term=NCT01384682&rank=1].) PMID:23596247

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

    Czech Academy of Sciences Publication Activity Database

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

    2003-01-01

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

  1. 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

    and incorporation of resistant forms in the long-lived CD4+ T cellular DNA compartment is not clear. Objective To investigate the relationship between lamivudine associated mutant-type 184V and the wild-type 184M proviral forms in the circulating CD4+ T cells of patients and low-level viremia. Study design Cross...... 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...... to only 8% in those with undetectable viremia. The presence of 184V was significantly associated with a higher viral load (P = 0.001). Patients with low-level viremia without 184V in the CD4+ T cellular DNA compartment, had a median plasma viral load of 135 copies/ml, while patients harbouring 184V had...

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

    International Nuclear Information System (INIS)

    Ma Shiliang; Sorensen, Annette Balle; Kunder, Sandra; Sorensen, Karina Dalsgaard; Quintanilla-Martinez, Leticia; Morris, David W.; Schmidt, Joerg; Pedersen, Finn Skou

    2006-01-01

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

  3. Correlation between LTR point mutations and proviral load levels among Human T cell Lymphotropic Virus type 1 (HTLV-1 asymptomatic carriers

    Directory of Open Access Journals (Sweden)

    Neto Walter K

    2011-12-01

    Full Text Available Abstract Background In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of Tax-responsive element (TRE of the genuine human T-cell leukemia virus type I (HTLV-1 viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1i nfected cells and alter the proviral load (PvL. To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden. Methods A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR and a PvL for Tax DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared. Results Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31, 27.5% (8/29, and 61.8% (21/34 of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both p p > 0.05. This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p p > 0. 05. Conclusions The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.

  4. Correlation between LTR point mutations and proviral load levels among human T cell lymphotropic virus type 1 (HTLV-1) asymptomatic carriers.

    Science.gov (United States)

    Neto, Walter K; Da-Costa, Antonio C; de Oliveira, Ana Carolina S; Martinez, Vanessa P; Nukui, Youko; Sabino, Ester C; Sanabani, Sabri S

    2011-12-13

    In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of Tax-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1-infected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden. A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR) and a PvL for Tax DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared. Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both p 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p 0. 05). The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.

  5. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA

    Directory of Open Access Journals (Sweden)

    Alex José Leite Torres

    2015-03-01

    Full Text Available Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.

  6. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA.

    Science.gov (United States)

    Torres, Alex José Leite; Brígido, Luis Fernando de Macedo; Abrahão, Marcos Herculano Nunes; Angelo, Ana Luiza Dias; de Jesus Ferreira, Gilcivaldo; Coelho, Luana Portes; Ferreira, João Leandro; Jorge, Célia Regina Mayoral Pedroso; Netto, Eduardo Martins; Brites, Carlos

    2015-01-01

    Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  7. DNA-cell conjugates

    Science.gov (United States)

    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.

  8. Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus

    Directory of Open Access Journals (Sweden)

    Fadrosh Douglas W

    2007-06-01

    Full Text Available Abstract Background Bracoviruses (BVs, a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV proviral genome. Results Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses. Conclusion Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s. We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of

  9. Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus.

    Science.gov (United States)

    Desjardins, Christopher A; Gundersen-Rindal, Dawn E; Hostetler, Jessica B; Tallon, Luke J; Fuester, Roger W; Schatz, Michael C; Pedroni, Monica J; Fadrosh, Douglas W; Haas, Brian J; Toms, Bradley S; Chen, Dan; Nene, Vishvanath

    2007-06-26

    Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome. Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses. Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to

  10. High incidence of proviral integrations in the Hoxa locus in a new model of E2a-PBX1-induced B-cell leukemia.

    Science.gov (United States)

    Bijl, Janet; Sauvageau, Martin; Thompson, Alexander; Sauvageau, Guy

    2005-01-15

    Relevant mouse models of E2a-PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a-PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3epsilon expression (CD3epsilon(-/-)). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a-PBX1 x CD3epsilon(-/-) compound transgenic animals is significantly accelerated when compared to control littermates, and document several known and novel integrations in these tumors. Of all common integration sites, a small region of 19 kb in the Hoxa gene locus, mostly between Hoxa6 and Hoxa10, represented 18% of all integrations in the E2a-PBX1 B-cell leukemia and was targeted in 86% of these leukemias compared to 17% in control tumors. Q-PCR assessment of expression levels for most Hoxa cluster genes in these tumors revealed an unprecedented impact of the proviral integrations on Hoxa gene expression, with tumors having one to seven different Hoxa genes overexpressed at levels up to 6600-fold above control values. Together our studies set the stage for modeling E2a-PBX1-induced B-cell leukemia and shed new light on the complexity pertaining to Hox gene regulation. In addition, our results show that the Hoxa gene cluster is preferentially targeted in E2a-PBX1-induced tumors, thus suggesting functional collaboration between these oncogenes in pre-B-cell tumors.

  11. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  12. Genotypic resistance test in proviral DNA can identify resistance mutations never detected in historical genotypic test in patients with low level or undetectable HIV-RNA.

    Science.gov (United States)

    Zaccarelli, Mauro; Santoro, Maria Mercedes; Armenia, Daniele; Borghi, Vanni; Gennari, William; Gori, Caterina; Forbici, Federica; Bertoli, Ada; Fabeni, Lavinia; Giannetti, Alberto; Cicalini, Stefania; Bellagamba, Rita; Andreoni, Massimo; Mastroianni, Claudio Maria; Mussini, Cristina; Ceccherini-Silberstein, Francesca; Perno, Carlo Federico; Antinori, Andrea

    2016-09-01

    Beyond the detection of resistant HIV strains found in plasma samples, archival HIV-DNA in peripheral blood mononuclear cells (PBMCs) might represent a reservoir of additional resistance. To characterize the HIV-1 resistance in PBMCs from patients with suppressed or low-level viremia (50-1000 copies/mL) and evaluate its added value compared to the resistance detected in previous plasma genotypic resistance tests (GRTs). HIV-1 infected patients selected for treatment change despite low/undetectable viremia were tested. Number and type of primary resistance mutations (PRMs) detected in PBMCs were compared to those detected in previous plasma GRTs. Logistic regression assessed factors associated with presence of at least one PRM in PBMCs. 468 patients with a PBMC GRT were analyzed; 149 of them had at least 2 plasma GRTs performed before PBMC genotyping. 42.3% of patients showed at least one PRM in PBMCs. The highest proportion of PRMs in PBMCs was observed for NRTI class (30.6%), followed by NNRTI (22.2%), PI (14.1%) and INI (4.9%). In 20.1% of patients, PRMs were detected only in PBMCs and not in any of the plasma GRT previously performed. By using multivariable analysis, a higher number of previous regimens, injecting drug-use route and a lower nadir CD4 were associated with significantly higher risk of detecting PRMs in PBMCs. Our findings support the usage of PBMC GRT in addition to the current recommended plasma RNA test, especially when therapeutic and/or resistance information is not available. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

    Science.gov (United States)

    Wayengera, Misaki

    2011-07-22

    LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines. 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.

  14. Cellular proviral HIV type 1 DNA load persists after long-term RT-inhibitor therapy in HIV type 1 infected persons

    NARCIS (Netherlands)

    Bruisten, S. M.; Reiss, P.; Loeliger, A. E.; van Swieten, P.; Schuurman, R.; Boucher, C. A.; Weverling, G. J.; Huisman, J. G.

    1998-01-01

    In a set of 42 antiretroviral naive HIV-1 infected persons who were treated with either Zidovudine (AZT) monotherapy, or a combination of AZT + ddC (Zalcitabine) or AZT + ddI (Didanosine), the HIV-1 DNA load was measured by competitive polymerase chain reaction (PCR) and related to the HIV-1 RNA

  15. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA

    OpenAIRE

    Torres, Alex José Leite; Brígido, Luis Fernando de Macedo; Abrahão, Marcos Herculano Nunes; Angelo, Ana Luiza Dias; Ferreira, Gilcivaldo de Jesus; Coelho, Luana Portes; Ferreira, João Leandro; Jorge, Célia Regina Mayoral Pedroso; Martins Netto, Eduardo; Brites, Carlos

    2015-01-01

    Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular m...

  16. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

    Directory of Open Access Journals (Sweden)

    Asunción Delgado

    2017-05-01

    Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

  17. Association between HLA Class I Alleles and Proviral Load in HTLV-I Associated Myelopathy/Tropical Spastic Paraperesis (HAM/TSP) Patients in Iranian Population.

    Science.gov (United States)

    Taghaddosi, Mahdi; Rezaee, S A Rahim; Rafatpanah, Houshang; Rajaei, Taraneh; Farid Hosseini, Reza; Narges, Valizadeh

    2013-03-01

    The aim of this study was to investigate the association between HLA class I alleles (HLA-A*02, HLA-A*24, HLA-Cw*08, HLA-B5401) and proviral load in HTLV-I associated myelopathy/tropical spastic paraperesis (HAM/TSP) patients in Iranian population. 20 new cases of HAM/TSP patients and 30 HTLV-I infected healthy carriers were recruited. Peripheral blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were isolated. DNA was extracted from PBMC.HTLV-I proviral load was calculated by Taqman quantitative real time polymerase chain reaction (qRT-PCR). PCR sequence-specific primer (PCR-SSP) reactions were performed to detect HLA-A, HLA-B and, HLA-Cw alleles. There was no significant difference in sex and age between asymptomatic and HAM/TSP group. The Mann-Whitney U test was used to compare proviral load between HAM/TSP patients and healthy carrier. Provirus load of HAM/TSP patients was significantly higher than that of HCs (P=0.003, Mann-Whitney U test).Odd ratio was calculated to determine association between class I alleles including (HLA-A*02, HLA-A*24, HLA-Cw*08) and risk of HAM/TSP development. We couldn't find any association between these class I alleles and risk of HAM/TSP development in our study. In our survey HLA-A*02, HLA-A24, HLA-Cw*08 didn't have protective effect on proviral load (P=0.075, P=0.060 and 0.650 Mann-Whitney U test respectively). In conclusion, certain HLA alleles with protective effect in one population may have not similar effect in other population. This may be because of pathogen polymorphism or host genetic heterogeneity and allele frequency in desired population.

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

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

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

  19. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

    Science.gov (United States)

    Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2017-05-09

    Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. High incidence of proviral integrations in the Hoxa locus in a new model of E2a–PBX1-induced B-cell leukemia

    OpenAIRE

    Bijl, Janet; Sauvageau, Martin; Thompson, Alexander; Sauvageau, Guy

    2005-01-01

    Relevant mouse models of E2a–PBX1-induced pre-B cell leukemia are still elusive. We now report the generation of a pre-B leukemia model using E2a–PBX1 transgenic mice, which lack mature and precursor T-cells as a result of engineered loss of CD3ε expression (CD3ε–/–). Using insertional mutagenesis and inverse-PCR, we show that B-cell leukemia development in the E2a–PBX1 × CD3ε–/– compound transgenic animals is significantly accelerated when compared to control littermates, and document severa...

  1. DNA repair , cell repair and radiosensitivity

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.

    1983-01-01

    Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

  2. 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.

  3. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    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

  4. 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

  5. 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.

  6. DNA repair in human cells

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

    1982-01-01

    Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

  7. Carga proviral do HTLV-1 e HTLV-2: um método simples através da PCR quantitativa em tempo real

    OpenAIRE

    Tamegão-Lopes,Bruna Pedroso; Rezende,Priscila Rocha; Maradei-Pereira,Luciana Maria Cunha; Lemos,José Alexandre Rodrigues de

    2006-01-01

    Os vírus linfotrópicos de células T humanas, quando integrados ao genoma da célula hospedeira, provírus, têm como marcador de replicação seu DNA proviral. A carga proviral parece ser um importante fator no desenvolvimento de patologias associadas a estes retrovírus. Neste estudo foi desenvolvida uma metodologia para quantificação absoluta da carga proviral dos HTLV-1 e HTLV-2 através da PCR em tempo real. Cinqüenta e três amostras de doadores de sangue com teste de ELISA reagente foram submet...

  8. Evaluation of HTLV-1 activity in HAM/TSP patients using proviral load and Tax mRNA expression after In Vitro lymphocyte activation.

    Science.gov (United States)

    Yari, Atefeh; Rezaee, Seyyed Abdolrahim; Valizadeh, Narges; Rajaee, Taraneh; Jazayeri, Seyyed Mohammad; Soltani, Mojdeh; Norouzi, Mehdi

    2014-07-01

    HTLV-1 is the first human retrovirus that has been recognized and is associated with HAM/TSP and ATLL. Studies have shown that less than five percent of HTLV-1 infected carriers develop HAM/TSP or ATLL and about ninety-five percent remain asymptomatic. Therefore, the proviral load with Tax may affect cellular genes such as cytokines and oncogenes, as well as involve in pathogenicity. Thirty HAM/TSP patients, thirty HTLV-1 healthy carriers, and MT-2 cell line were evaluated for HTLV-1 activity. PBMCs were isolated and activated using PMA and ionomycine. Real-time PCR and TaqMan methods were performed using specific primers and fluorescence probes for Tax expression and proviral load assessment. B2microglobulin (β2m) and albumin were used as controls in Tax expression and in proviral load, respectively. An insignificant increase in Tax expression was observed in rest PBMCs of HAM/TSP patients compared to healthy carriers. However, after lymphocyte activation there was a significant increase in Tax expression in HAM/TSP patients (P=0.042). The Proviral load in patients was significantly higher than in carriers. Moreover, there was a significant correlation between Tax mRNA expression in activated PBMCs and proviral load (R=0.37, P=0.012). Although proviral load had been addressed as a valuable index for monitoring HTLV-1 infected subjects, the results of this study demonstrated that Tax expression in activated PBMCs along with proviral load assessment in HAM/TSP patients are a more reliable factor for determining the prognosis and monitoring healthy carriers and HAM/TSP patients.

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

    Directory of Open Access Journals (Sweden)

    Chieia Marco

    2008-07-01

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

  10. DNA replication and repair in Tilapia cells

    International Nuclear Information System (INIS)

    Yew, F.H.; Chang, L.M.

    1984-01-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

  11. Instructing cells with programmable peptide DNA hybrids

    Science.gov (United States)

    Freeman, Ronit; Stephanopoulos, Nicholas; Álvarez, Zaida; Lewis, Jacob A; Sur, Shantanu; Serrano, Chris M; Boekhoven, Job; Lee, Sungsoo S.; Stupp, Samuel I.

    2017-01-01

    The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed. PMID:28691701

  12. Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals.

    Science.gov (United States)

    Trejbalová, Kateřina; Kovářová, Denisa; Blažková, Jana; Machala, Ladislav; Jilich, David; Weber, Jan; Kučerová, Dana; Vencálek, Ondřej; Hirsch, Ivan; Hejnar, Jiří

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR. In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation. Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.

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

    International Nuclear Information System (INIS)

    Dickie, Peter; Roberts, Amanda; Uwiera, Richard; Witmer, Jennifer; Sharma, Kirti; Kopp, Jeffrey B.

    2004-01-01

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

  14. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1981-01-01

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

  15. DNA synthesis in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Painter, R.B.; California Univ., San Francisco; Young, B.R.

    1987-01-01

    One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v. -induced inhibition of replicon initiation. (author

  16. Prenatal Cell-Free DNA Screening

    Science.gov (United States)

    ... poses no physical risks for you or your baby. While prenatal cell-free DNA screening might cause anxiety, it might help you avoid the need for more invasive tests, treatment or monitoring during your pregnancy. Keep in mind, however, that ...

  17. Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

    Directory of Open Access Journals (Sweden)

    Tianhong Su

    2018-03-01

    Full Text Available Mitochondrial DNA (mtDNA mutations accumulate in somatic stem cells during ageing and cause mitochondrial dysfunction. In this review, we summarize the studies that link mtDNA mutations to stem cell ageing. We discuss the age-related behaviours of the somatic mtDNA mutations in stem cell populations and how they potentially contribute to stem cell ageing by altering mitochondrial properties in humans and in mtDNA-mutator mice. We also draw attention to the diverse fates of the mtDNA mutations with different origins during ageing, with potential selective pressures on the germline inherited but not the somatic mtDNA mutations.

  18. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-01

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

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

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

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

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Maria T. V. Romanos

    2011-04-01

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

  4. Extrachromosomal HIV-1 DNA in persistently infected U937 cells.

    Science.gov (United States)

    Pauza, C D; Singh, M K

    1990-08-01

    Persistent human immunodeficiency virus type 1 (HIV-1) infection of U937 monocytic cells resulted in the accumulation of novel forms of extrachromosomal viral DNA. These DNA species are larger than the genome size of HIV-1 and persist indefinitely. The extrachromosomal viral DNA species (E-DNA) were shown to be structurally stable by subcloning of infected cell lines and restriction fragment analysis. Similar E-DNA structures were observed in independent infections. Persistently infected monocytic cells had low levels of viral antigens, reflecting the low levels of viral RNA that were detected. These results support a role for E-DNA in persistent HIV-1 infection of monocytic cells.

  5. Oxidized Extracellular DNA as a Stress Signal in Human Cells

    Directory of Open Access Journals (Sweden)

    Aleksei V. Ermakov

    2013-01-01

    Full Text Available The term “cell-free DNA” (cfDNA was recently coined for DNA fragments from plasma/serum, while DNA present in in vitro cell culture media is known as extracellular DNA (ecDNA. Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2 that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments.

  6. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

    International Nuclear Information System (INIS)

    Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

    2005-01-01

    Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

  7. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  8. Isolated HeLa cell nuclei synthesize meaningful DNA.

    OpenAIRE

    Kristensen, T; Prydz, H

    1985-01-01

    DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The resu...

  9. Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells.

    Science.gov (United States)

    Hyun, Sun-Yi; Rosen, Eliot M; Jang, Young-Joo

    2012-07-06

    DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Evaluation of HTLV-1 HBZ and proviral load, together with host IFN λ3, in pathogenesis of HAM/TSP.

    Science.gov (United States)

    Mozhgani, Sayed-Hamidreza; Jaberi, Najmeh; Rezaee, Seyed Abdolrahim; Bustani, Reza; Jazayeri, Seyed Mohammad; Akbarin, Mohammad Mehdi; Milani, Saeideh; Tarokhian, Hanieh; Norouzi, Mehdi

    2017-06-01

    Human T-cell lymphotropic virus 1 (HTLV-1) is associated with two progressive diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). Although HTLV-1 proviral load (PVL) has been introduced as a risk factor for these diseases' progression, it is not sufficient on its own to yield an accurate estimation of the outcome of the infection. In the present study, PVL and HTLV-1 basic leucine zipper factor (HBZ) expression level as viral factors, and IFN λ3 as a host factor, were evaluated in HAM/TSP patients and HTLV-1 asymptomatic carriers (ACs). During 2014-2015, 12 HAM/TSP patients and 18 ACs who had been referred to the HTLV-1 Clinic, Ghaem Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran, were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and the DNA and mRNA were extracted for quantification of HBZ, IFN λ3 expression, and PVL using real-time PCR (TaqMan method). Although the PVL was higher in the HAM/TSP group, with a 94% confidence interval, there were no considerable differences in terms of HBZ mRNA and PVL between ACs and HAM patients. IFN λ3 expression in the HAM/TSP group was significantly higher than in the ACs (P = 0.02). To the best of our knowledge, no study has evaluated the expression level of IFN λ3 in HTLV-1 positive patients. The immune response against HTLV-1 viral antigens and virulent factors will therefore further refine our knowledge of interactions between the virus and host in the pathogenesis of HTLV-1-related disorders. The virus PVL and the host IFN λ3 can be used as pathogenic factors of HTLV-1 infected patients at risk of HAM/TSP manifestation. J. Med. Virol. 89:1102-1107, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. 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 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.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.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 with fetal chromosomal aneuploidy, regardless of fetal

  12. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

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

    Directory of Open Access Journals (Sweden)

    Katarina Znidar

    2016-01-01

    Full Text Available 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.

  14. 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.

  15. Treatment of cancer cells with methioninase produces DNA hypomethylation and increases DNA synthesis.

    Science.gov (United States)

    Machover, David; Zittoun, Jacqueline; Saffroy, Raphaël; Broët, Philippe; Giraudier, Stéphane; Magnaldo, Thierry; Goldschmidt, Emma; Debuire, Brigitte; Orrico, Mireille; Tan, Yuying; Mishal, Zohar; Chevallier, Odile; Tonetti, Carole; Jouault, Hélène; Ulusakarya, Ayhan; Tanguy, Marie-Laure; Metzger, Gérard; Hoffman, Robert M

    2002-08-15

    Methionine depletion in the human cell line CCRF-CEM through the action of recombinant methioninase (rMETase), a methionine-cleaving enzyme, was previously demonstrated to produce a strong cytotoxic synergistic effect with fluorouracil (FUra) throughout a broad range of concentrations of FUra and rMETase, including subcytotoxic levels of rMETase. Potentiation was associated with a decrease in free thymidylate synthase from preexisting levels. To further investigate the action of rMETase on CCRF-CEM cells, in the present study we explored the effects of rMETase as a single agent on DNA methylation levels and DNA synthesis, which may be changed as a result of deprivation of methionine. Cells treated with rMETase under subcytotoxic conditions contained significantly lower levels of genomic methylated DNA than did control cells, as demonstrated by incorporation of the methyl radical of [methyl-(3)H]S-adenosylmethionine in DNA and by use of methylation-sensitive arbitrarily primed PCR. DNA hypomethylation produced by rMETase was of similar magnitude as that produced with the DNA methyltransferase inhibitor 5-azacytidine. Cells exposed to rMETase synthesized significantly more DNA than did untreated cells. Incorporation of [6-3H]thymidine and [6-3H]2'-deoxyuridine in these cells was augmented over that in control by mean factors of 1.78 and 2.36, respectively. Increased 3H nucleoside incorporation resulted in greater numbers of nuclear grains as demonstrated by autoradiography. The increase in DNA synthesis induced by rMETase is likely to result from enhancement of DNA repair because it was not accompanied by differences in cell cycle phase distribution or in total DNA content as determined by flow cytometry. We hypothesize that potentiation of FUra cytotoxicity by rMETase may result from increased inhibition of thymidylate synthase, together with DNA hypomethylation and enhanced DNA repair that could be involved in cell responses to drug-induced damage.

  16. Uptake of DNA by cancer cells without a transfection reagent.

    Science.gov (United States)

    Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

    2017-01-21

    Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting

  17. Induced pluripotent stem cells with a mitochondrial DNA deletion.

    Science.gov (United States)

    Cherry, Anne B C; Gagne, Katelyn E; McLoughlin, Erin M; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D; Zhang, Jin; Zon, Rebecca L; Ince, Tan A; Neufeld, Ellis J; Lerou, Paul H; Fleming, Mark D; Daley, George Q; Agarwal, Suneet

    2013-07-01

    In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here, we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. Copyright © 2013 AlphaMed Press.

  18. Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

    Science.gov (United States)

    Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

    2013-01-01

    In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

  19. 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.

  20. 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.

  1. High HTLV-1 proviral load, a marker for HTLV-1 associated myelopathy/tropical spastic paraparesis, is also detected in patients with infective dermatitis associated with HTLV-1

    Directory of Open Access Journals (Sweden)

    J. Primo

    2009-08-01

    Full Text Available Salvador (BA, Brazil is an endemic area for human T-cell lymphotrophic virus type 1 (HTLV-1. The overall prevalence of HTLV-1 infection in the general population has been estimated to be 1.76%. HTLV-1 carriers may develop a variety of diseases such as adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP and infective dermatitis associated with HTLV-1 (IDH. IDH is a chronic and severe form of childhood exudative and infective dermatitis involving mainly the scalp, neck and ears. It has recently been observed that 30% of patients with IDH develop juvenile HAM/TSP. The replication of HTLV-1 has been reported to be greater in adult HAM/TSP patients than in asymptomatic HTLV-1 carriers. In the current study, the proviral load of 28 children and adolescents with IDH not associated with HAM/TSP was determined and the results were compared to those obtained in 28 HTLV-1 adult carriers and 28 adult patients with HAM/TSP. The proviral load in IDH patients was similar to that of patients with HAM/TSP and much higher than that found in HTLV-1 carriers. The high levels of proviral load in IDH patients were not associated with age, duration of illness, duration of breast-feeding, or activity status of the skin disease. Since proviral load is associated with neurological disability, these data support the view that IDH patients are at high risk of developing HAM/TSP.

  2. Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

    Science.gov (United States)

    Paradkar, Prasad N; Duchemin, Jean-Bernard; Rodriguez-Andres, Julio; Trinidad, Lee; Walker, Peter J

    2015-09-01

    Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

  3. Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

    Directory of Open Access Journals (Sweden)

    Prasad N Paradkar

    2015-09-01

    Full Text Available Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

  4. Cell-Free DNA in Metastatic Colorectal Cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise G; Boysen, Anders K; Pallisgård, Niels

    2017-01-01

    -analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. MATERIALS AND METHODS: A systematic literature search of PubMed and Embase was performed by two...... therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients...... with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment....

  5. HTLV-1 proviral load in cerebrospinal fluid may not be a good marker to differentiate asymptomatic carriers with high proviral load in blood from HAM/TSP patients.

    Science.gov (United States)

    Martins, Marina Lobato; de Freitas Carneiro-Proietti, Anna Bárbara; Nicolato, Rodrigo; de Miranda, Débora Marques; Romanelli, Luiz Cláudio Ferreira

    2018-03-27

    An elevated human T cell leukemia virus type 1 (HTLV-1) proviral load (PVL) is an important risk factor for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), although there is a considerable frequency of asymptomatic carriers (AC) with high PVL in blood. Our objective was to evaluate whether PVL quantified in cerebrospinal fluid (CSF) is helpful to distinguish AC from HAM when AC have high PVL in blood (AC H ). AC H (n = 7) were characterized to have high PVL in blood by quantification of samples collected over time (mean 7 years). HAM patients (n = 14) also had analyzed blood samples collected at different times (mean 9 years). Comparing paired CSF and blood samples of each individual, CSF PVL mean was 4.7-fold higher than blood PVL in the AC H group and 10.8-fold in the HAM group. CSF PVL was significantly greater than blood PVL in the HAM group (p = 0.004), but not in the AC H group. Important to highlight, CSF PVL was not significantly different between the AC H and the HAM groups. These results suggested that significantly higher PVL in CSF than in blood is a hallmark of HAM/TSP patients, but this is also true for asymptomatic carriers with high PVL in blood, thus reducing its usefulness as a marker for HAM/TSP. A greater number of AC H should be analyzed, but whether they will eventually develop HAM/TSP or why they have not developed the disease are still questions to be clarified. Longitudinal studies are necessary to answer these questions.

  6. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. [Los Alamos National Lab., NM (United States); Chen, D.S. [Rochester Univ., NY (United States). Dept. of Radiation Oncology

    1993-02-01

    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.

  7. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. (Los Alamos National Lab., NM (United States)); Chen, D.S. (Rochester Univ., NY (United States). Dept. of Radiation Oncology)

    1993-01-01

    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.

  8. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-01-01

    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

  9. Cell size and the initiation of DNA replication in bacteria.

    Directory of Open Access Journals (Sweden)

    Norbert S Hill

    Full Text Available In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.

  10. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    Science.gov (United States)

    Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell Iv, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

    2017-07-01

    Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  11. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  12. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

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

    2013-01-01

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

  13. 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. DNA replication and repair of Tilapia cells: Pt. 2

    International Nuclear Information System (INIS)

    Chen, J.D.; Yew, F.H.

    1988-01-01

    TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 0 C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 0 C to the sublethal high temperature of 37 0 C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37 0 C the rate of DNA synthesis overshot the 31 0 C control level by 180%. When moved to low temperature (18 0 C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25 0 C, but not at 18 0 C. Initiation of nascent DNA synthesis was blocked at 4Jm -2 in TO-2 cells compared with ≤ 1Jm -2 in mammalian cells. After 9Jm -2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair. (author)

  15. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  16. Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection.

    Science.gov (United States)

    Gillet, Nicolas A; Gutiérrez, Gerónimo; Rodriguez, Sabrina M; de Brogniez, Alix; Renotte, Nathalie; Alvarez, Irene; Trono, Karina; Willems, Luc

    2013-01-01

    Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed

  17. Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection.

    Directory of Open Access Journals (Sweden)

    Nicolas A Gillet

    Full Text Available Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1 and bovine leukemia virus (BLV induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle or via clonal expansion of the infected cells (mitotic cycle. The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone. We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1 initial integration inside genes or promoters and (2 host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in

  18. 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.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  20. Decreased stability of DNA in cells treated with alkylating agents

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Cedars Medical Center, Miami, FL (United States))

    1990-12-01

    A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

  1. 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...

  2. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... ... High Sensitivity Bioanalyzer Chip (Agilent) to check for quality and size distribution. DNA library frag- ments were diluted, denatured and hybridized to a lawn of oligonucleotides immobilized on the flow-cell surface. Hy- bridized DNA template was amplified using immobilized oligonucleotides as primers.

  3. DNA synthesis in permeabilized WI38 and MRC5 cells

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Carpenter, J.G.

    1980-01-01

    DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

  4. DNA Charge Transport: From Chemical Principles to the Cell

    Science.gov (United States)

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

    2016-01-01

    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

  5. Increment of DNA topoisomerases in chemically and virally transformed cells

    International Nuclear Information System (INIS)

    Crespi, M.D.; Mladovan, A.G.; Baldi, A.

    1988-01-01

    The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo[a]pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between 32 P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo[a]pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA

  6. 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.

  7. Circulating cell free DNA as a predictor of systemic lupus ...

    African Journals Online (AJOL)

    Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy. Olfat M. Hendy, Tawfik Abdel Motalib, Mona A. El Shafie, Fatma A. Khalaf, Sobhy E. Kotb, Aziza Khalil, Salwa R. Ali ...

  8. DNA Based Electrochromic and Photovoltaic Cells

    Science.gov (United States)

    2012-01-01

    performed in the Prof. Dr. Ana Flavia Nogueira (UNICAMP) labs and now this investigation is continued. 200 300 400 500 600 700 800 900 1000 1100 0 10...with DNA developed in Prof. Dr. Ana Flavia Nogueira (UNICAMP-Brazil) laboratory [the data are extracted from the reference 30]. The small solar...by Prof. Dr. Ana Flavia Nogueira from DQ-UNICAMP revealed increase of the solar efficiency after DNA application over the TiO2 thin film sensitized

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

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

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

  10. Cell-Free DNA Screening for Aneuploidy and Microdeletion Syndromes.

    Science.gov (United States)

    Shaffer, Brian L; Norton, Mary E

    2018-03-01

    Cell-free DNA (cfDNA) screening for the common aneuploidies is an accurate noninvasive screen for the common autosomal and sex chromosome aneuploidies. However, cfDNA screening should not be considered a diagnostic test, and the positive predictive value should be used in counseling women with a positive test regarding the option for diagnostic testing. Compared with traditional screening, cfDNA may not detect as many chromosomal abnormalities of importance. Furthermore, due to the low prevalence of recurrent copy number variants, the clinical utility in screening for microdeletions and duplications is uncertain and is not recommended for the general obstetric population. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 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.

  12. Immunmodulation of the Th cell differentiation using DNA immunization

    OpenAIRE

    Muzzulini, Till

    2010-01-01

    Th cells regulate the immune response in part by the secretion of cytokines. Upon stimulation with antigen naive Th cells differentiate. During this differentiation they receive an imprinting for a certain cytokine profile. It depends on this imprint whether the immune response is adequate or pathologic i.e. autoimmune. Therefore the manipulation of this differentiation is a possibility to treat autoimmunity. This manipulation can be achieved through DNA immunisation. In DNA immunisation simp...

  13. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    Science.gov (United States)

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  14. Cell-Free DNA and Active Rejection in Kidney Allografts.

    Science.gov (United States)

    Bloom, Roy D; Bromberg, Jonathan S; Poggio, Emilio D; Bunnapradist, Suphamai; Langone, Anthony J; Sood, Puneet; Matas, Arthur J; Mehta, Shikha; Mannon, Roslyn B; Sharfuddin, Asif; Fischbach, Bernard; Narayanan, Mohanram; Jordan, Stanley C; Cohen, David; Weir, Matthew R; Hiller, David; Prasad, Preethi; Woodward, Robert N; Grskovic, Marica; Sninsky, John J; Yee, James P; Brennan, Daniel C

    2017-07-01

    Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls ( P =0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection. Copyright © 2017 by the American Society of Nephrology.

  15. Nuclear DNA Content Varies with Cell Size across Human Cell Types

    Science.gov (United States)

    Gillooly, James F.; Hein, Andrew; Damiani, Rachel

    2015-01-01

    Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity. PMID:26134319

  16. Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1979-01-01

    Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

  17. In vitro infection of human epidermal Langerhans' cells with HIV-1.

    Science.gov (United States)

    Ramazzotti, E; Marconi, A; Re, M C; Girolomoni, G; Cenacchi, G; Vignoli, M; Zambruno, G; Furlini, G; La Placa, M; Giannetti, A

    1995-05-01

    Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LC from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LC (10-25% of CD1a+/CD4+ cells), deprived of contaminating T cells and then incubated with HIV-1IIIB. After 24 hr, purified LC and LC-depleted EC fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PCR) analysis showed the presence of HIV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LC, LC-depleted EC or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1IIIB for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Co-cultures of C8166 cells with purified LC or LC-enriched EC previously exposed to HIV-1IIIB exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.

  18. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    Science.gov (United States)

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    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 (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. PMID:24722772

  19. DNA mismatch repair and its many roles in eukaryotic cells

    DEFF Research Database (Denmark)

    Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

    2017-01-01

    in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays......DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers...... novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore...

  20. DNA mismatch repair and its many roles in eukaryotic cells.

    Science.gov (United States)

    Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

    2017-07-01

    DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1-independent subpathways of MMR is not known. This review summarizes recent literature on eukaryotic MMR, with emphasis on the diverse cellular roles of eukaryotic MMR proteins, the mechanism of strand discrimination and cross-talk/interactions between and co-regulation of MMR and other DNA repair pathways in eukaryotic cells. The main conclusion of the review is that MMR proteins contribute to genome stability through their ability to recognize and promote an appropriate cellular response to aberrant DNA structures, especially when they arise during DNA replication. Although the molecular mechanism of MMR in the eukaryotic cell is still not completely understood, increased used of single-molecule analyses in the future may yield new insight into these unsolved questions. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior

    Science.gov (United States)

    Kearney, Cathal J.; Lucas, Christopher R.; O'Brien, Fergal J.; Castro, Carlos E.

    2016-01-01

    DNA origami is a DNA-based nanotechnology that utilizes programmed combinations of short complementary oligonucleotides to fold a large single strand of DNA into precise 2-D and 3-D shapes. The exquisite nanoscale shape control of this inherently biocompatible material is combined with the potential to spatially address the origami structures with diverse cargos including drugs, antibodies, nucleic acid sequences, small molecules and inorganic particles. This programmable flexibility enables the fabrication of precise nanoscale devices that have already shown great potential for biomedical applications such as: drug delivery, biosensing and synthetic nanopore formation. In this Progress Report, we will review the advances in the DNA origami field since its inception several years ago and then focus on how these DNA-nanodevices can be designed to interact with cells to direct or probe their behavior. PMID:26840503

  2. Segrosome complex formation during DNA trafficking in bacterial cell division

    Directory of Open Access Journals (Sweden)

    Maria A. Oliva

    2016-09-01

    Full Text Available 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 specialised 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.

  3. Urea selectively induces DNA synthesis in renal epithelial cells.

    Science.gov (United States)

    Cohen, D M; Gullans, S R

    1993-04-01

    Hyperosmotic stress with the functionally impermeant solute NaCl has been shown by us and others to inhibit cell growth and DNA synthesis. Several lines of evidence suggest that urea, the other principal renal medullary solute, may exert a growth-promoting effect on renal epithelial cells. Among these is the finding that urea upregulates expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos. In the present study, urea, in concentrations characteristic of the renal medulla, increased [3H]thymidine incorporation approximately threefold in confluent, growth-suppressed Madin-Darby canine kidney (MDCK) cells, whereas another readily membrane-permeant solute, glycerol, did not. Urea also overcame the inhibitory effect of hyperosmotic NaCl on DNA synthesis. The urea-induced increase in [3H]thymidine incorporation was also evident in the renal epithelial LLC-PK1 cell line, but not in renal nonepithelial and epithelial nonrenal cell types examined. In addition, it was associated with a 15% increase in total DNA content measured fluorometrically at 24 h of treatment. There was, however, no associated increase in cell proliferation as measured by cell number, total protein content, or cell cycle distribution. Urea also failed to induce polyploidy or aneuploidy. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased DNA synthesis through an undefined and potentially novel mechanism.

  4. DNA ligase III is involved in a DNA-PK independent pathway of NHEJ in human cells

    International Nuclear Information System (INIS)

    Wang, H.; Perrault, A.R.; Qin, W.; Wang, H.; Iliakis, G.

    2003-01-01

    Full text: Double strand breaks (DSB) induced by ionizing radiation (IR) and other cytotoxic agents in the genome of higher eukaryotes are thought to be repaired either by homologous recombination repair (HRR), or non-homologous endjoining (NHEJ). We previously reported the operation of two components of NHEJ in vivo: a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK independent component that acts as a backup (basic or B-NHEJ) and operates with kinetics an order of magnitude slower. To gain further insight into the mechanisms of B-NHEJ, we investigated DNA endjoining in extracts 180BR, a human cell line deficient in DNA ligase IV, using an in vitro plasmid-based DNA endjoining assay. An anti DNA ligase III antibody inhibited almost completely DNA endjoining activity in these extracts. On the other hand, an anti DNA ligase I antibody had no measurable effect in DNA endjoining activity. Immunodepletion of DNA ligase III from 180BR cell extracts abolished the DNA endjoining activity, which could be restored by addition of purified human DNA ligase IIIb. Full-length DNA ligase III bound to double stranded DNA and stimulated DNA endjoining in both intermolecular and intramolecular ligation. Furthermore, fractionation of HeLa cell extracts demonstrated the presence of an activity stimulating the function of DNA ligase III. Based on these observations we propose that DNA ligase III is the ligase operating in B-NHEJ

  5. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

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

    Science.gov (United States)

    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 oligonucleotides within cell nuclei. An oligonucleotide–psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei. PMID:8990164

  7. 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

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

    International Nuclear Information System (INIS)

    Sestili, Piero; Paolillo, Marco; Lenzi, Monia; Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara; Fimognari, Carmela

    2010-01-01

    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 μM 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 of

  9. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    International Nuclear Information System (INIS)

    Huselton, C.A.; Hill, H.Z.

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals

  10. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    Energy Technology Data Exchange (ETDEWEB)

    Huselton, C.A.; Hill, H.Z. (New Jersey Medical School, Newark (USA))

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals.

  11. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  12. Chromatin factors affecting DNA repair in mammalian cell nuclei

    International Nuclear Information System (INIS)

    Harless, J.; Hittelman, W.; Meyn, R.; Hewitt, R.

    1983-01-01

    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

  13. Proviral Structure, Chromosomal Location, and Expression of HERV-K-T47D, a Novel Human Endogenous Retrovirus Derived from T47D Particles

    OpenAIRE

    Seifarth, Wolfgang; Baust, Corinna; Murr, Andreas; Skladny, Heyko; Krieg-Schneider, Frank; Blusch, Jürgen; Werner, Thomas; Hehlmann, Rüdiger; Leib-Mösch, Christine

    1998-01-01

    We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408–6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome compris...

  14. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    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)

  15. UV-induced DNA repair in leukemic cell differentiation

    International Nuclear Information System (INIS)

    Nakamaki, Tsuyoshi; Sakashita, Akiko; Tomoyasu, Shigeru; Tsuruoka, Nobuyoshi; Ajiri, Teizo.

    1989-01-01

    Ultraviolet light (UV)-induced DNA repair during myeloid leukemic cell differentiation was examined. Human myeloid leukemic cells could be induced to differentiate in vitro into mature cells by various chemical inducers that lost their proliferating potencies. In spite of decrease of proliferation capacity, almost all these terminally differentiated myeloid leukemic cells invariably showed UV-induced unscheduled DNA synthesis (UDS) at low energy of UV irradiation (3-5 J/m 2 ). This indicated that the terminally differentiated myeloid leukemic cells are functionally quite different from mature granulocytes in chronic myeloid leukemia (CML) or in normal peripheral blood. In HL-60 cells, UV-survival was enhanced in the process of differentiation induced by 1.25% DMSO or 0.6 mM sodium n-butyrate. The degree of enhancement of UV-survival was correlated with the increased amount of UDS. The process of myeloid leukemic cell differentiation which is completed without loss of capacity performing repair DNA synthesis was one of the characteristics of the terminally differentiated myeloid leukemic cells induced by chemical inducers in vitro and this function may support the hypothesis that DNA breaking and rejoining are involved in a mechanism of cytodifferentiation. (author)

  16. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could...

  17. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

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

  18. scid cells efficiently integrate hairpin and linear DNA substrates.

    Science.gov (United States)

    Staunton, J E; Weaver, D T

    1994-01-01

    The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination. Images PMID:8196630

  19. Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing.

    Science.gov (United States)

    Vaca-Paniagua, Felipe; Oliver, Javier; Nogueira da Costa, Andre; Merle, Philippe; McKay, James; Herceg, Zdenko; Holmila, Reetta

    2015-01-01

    To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

  20. 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

  1. Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

    International Nuclear Information System (INIS)

    Singh, Rana P.

    2017-01-01

    Solid tumors are mostly treated with radiotherapy. Radiotherapy is toxic to normal tissues and also promote the invasiveness and radioresistance in cancer cells. The resistance against radiotherapy and adverse effects to normal cells reduce the overall therapeutic effects of the treatment. Radiosensitizing agents usually show limited success during clinical trials. Therefore, the search and development of new radiosensitizers showing selective response to only cancer cells is desirable. We analyzed the radiosensitizing effects including cell death effect of silibinin, a phytochemical on prostate cancer cells. Silibinin enhanced gamma radiation (2.5-10 Gy) induced inhibition in colony formation selectively in prostate cancer cells. In cell cycle progression, G2/M phase is the most sensitive phase for radiation-induced damage which was delayed by the compound treatment in radiation exposed cells. The lower concentrations of silibinin substantially enhanced radiation-induced apoptosis. A prolonged reactive oxygen species production was also observed in these treatments EGFR signaling pathway can contribute to radiation-induced pro-survival mechanisms and to the therapeutic resistance. Agent treatment reduced the IR-induced EGFR phosphorylation and consequently reversed the resistance mediating mechanisms within the cancer cell. Thus, inhibiting DNA repair in cancer cells would enhance therapeutic response of radiation in cancer cells. Silibinin affected the localization of EGFR and DNA-dependent protein kinase, the DNA-PK is known to be an important mediator of DSB repair in human cells, and showed increased number of pH2AX (ser139) foci, and thus indicating lower DNA repair in these cancer cells. This was also confirmed in the tumor xenograft study. Our findings suggest that a combination of silibinin with radiation could be an effective treatment of radioresistant human prostate cancer and warrants further investigation. (author)

  2. 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....

  3. BACTERIAL CELL KILLING MEDIATED BY TOPOISOMERASE I DNA CLEAVAGE ACTIVITY

    Science.gov (United States)

    Cheng, Bokun; Shukla, Shikha; Vasunilashorn, Sarinnapha; Mukhopadhyay, Somshuvra; Tse-Dinh, Yuk-Ching

    2005-01-01

    DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. PMID:16159875

  4. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  5. [Genetic transformation and fate of heterological DNA in bacterial cells].

    Science.gov (United States)

    Piechowska, Mirosława

    2015-01-01

    Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

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

    International Nuclear Information System (INIS)

    Mohamed, R.; Lavin, M.F.

    1986-01-01

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

  7. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    Park, Sun Young; Choi, Bongkun; Cheon, Hwanju; Pak, Youngmi Kim; Kulawiec, Mariola; Singh, Keshav K.; Lee, Myung-Shik

    2004-01-01

    We have reported that mitochondrial DNA-depleted ρ 0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ 0 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 ρ 0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ 0 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 ρ 0 cells. These results support the mitochondrial theory of aging, and suggest that ρ 0 cells could serve as an in vitro model for aged cells

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

    International Nuclear Information System (INIS)

    Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

    2012-01-01

    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

  9. 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.

  10. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs.

  11. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

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

  12. 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.

  13. Multi-scale Modeling of Chromosomal DNA in Living Cells

    Science.gov (United States)

    Spakowitz, Andrew

    The organization and dynamics of chromosomal DNA play a pivotal role in a range of biological processes, including gene regulation, homologous recombination, replication, and segregation. Establishing a quantitative theoretical model of DNA organization and dynamics would be valuable in bridging the gap between the molecular-level packaging of DNA and genome-scale chromosomal processes. Our research group utilizes analytical theory and computational modeling to establish a predictive theoretical model of chromosomal organization and dynamics. In this talk, I will discuss our efforts to develop multi-scale polymer models of chromosomal DNA that are both sufficiently detailed to address specific protein-DNA interactions while capturing experimentally relevant time and length scales. I will demonstrate how these modeling efforts are capable of quantitatively capturing aspects of behavior of chromosomal DNA in both prokaryotic and eukaryotic cells. This talk will illustrate that capturing dynamical behavior of chromosomal DNA at various length scales necessitates a range of theoretical treatments that accommodate the critical physical contributions that are relevant to in vivo behavior at these disparate length and time scales. National Science Foundation, Physics of Living Systems Program (PHY-1305516).

  14. 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.

  15. DNA Translocation and Cell Electroporation in Micro and Nanofluidic Devices

    Science.gov (United States)

    Gupta, Cherry

    The cell membrane is made of a thin (˜ 5nm) lipid bilayer which serves as an effective insulator and diffusion barrier for entities external to the cell from entering the cell. However, for research, diagnostic and therapeutic purposes, there is a need to deliver molecules of interest to the interior of live cells. This is usually accomplished by two methods: (a) carrier mediated delivery which consists of encapsulating the gene/molecule of interest either in an empty viral capsid or in chemically formulated lipoplex or polyplex nanoparticles, or (b) physical methods of delivery, which include the use of different kinds of forces to create reversible pores on the cell membrane (sonoporation, electroporation) or directly inject molecules to the cell cytosol (Gene Gun, microinjection). Of the aforementioned techniques, electroporation is the most commonly used due to it simplicity and ease of use. Our group recently demonstrated a nanochannel based electroporation (NEP) technique, in which two microchannels (˜40 m diameter) are connected by a nanochannel (˜ 200-400 mum diameter) in the center. A cell is positioned in one microchannel such that it makes contact with the nanochannel and transfection agents are placed in the other microchannel. An external electric field applied across the device locally porates the cell where it touches the nancohannel and drives the transfection agents into the cell. Besides maintaining high cell viability and achieving dose control, an important feature of NEP is the delivery of large molecules such as plasmids and quantum dots directly into the cell cytosol. In contrast, delivery of large plasmids during bulk electroporation, wherein cells and genes/plasmids are mixed in a buffered medium and an external electric field is applied across the mixture which electroporates the cells, is via formation of cell membrane bound aggregates which get endocytosed post pulsation. Various mechanisms of DNA transport across the membrane have

  16. Blood Cell Mitochondrial DNA Content and Premature Ovarian Aging

    Science.gov (United States)

    Cacciatore, Chiara; Busnelli, Marta; Rossetti, Raffaella; Bonetti, Silvia; Paffoni, Alessio; Mari, Daniela; Ragni, Guido; Persani, Luca; Arosio, M.; Beck-Peccoz, P.; Biondi, M.; Bione, S.; Bruni, V.; Brigante, C.; Cannavo`, S.; Cavallo, L.; Cisternino, M.; Colombo, I.; Corbetta, S.; Crosignani, P.G.; D'Avanzo, M.G.; Dalpra, L.; Danesino, C.; Di Battista, E.; Di Prospero, F.; Donti, E.; Einaudi, S.; Falorni, A.; Foresta, C.; Fusi, F.; Garofalo, N.; Giotti, I.; Lanzi, R.; Larizza, D.; Locatelli, N.; Loli, P.; Madaschi, S.; Maghnie, M.; Maiore, S.; Mantero, F.; Marozzi, A.; Marzotti, S.; Migone, N.; Nappi, R.; Palli, D.; Patricelli, M.G.; Pisani, C.; Prontera, P.; Petraglia, F.; Radetti, G.; Renieri, A.; Ricca, I.; Ripamonti, A.; Rossetti, R.; Russo, G.; Russo, S.; Tonacchera, M.; Toniolo, D.; Torricelli, F.; Vegetti, W.; Villa, N.; Vineis, P.; Wasniewsk, M.; Zuffardi, O.

    2012-01-01

    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. PMID:22879975

  17. Circulating Cell-Free DNA Differentiates Severity of Inflammation.

    Science.gov (United States)

    Frank, Mayu O

    2016-10-01

    As the U.S. population ages, the incidence of chronic disease will rise. Chronic diseases have been linked to chronic inflammation. The purpose of this review is to summarize the literature on cell-free DNA (cfDNA) in relation to inflammation. PubMed, EMBASE, and Web of Science were searched. Inclusion criteria were noninterventional studies on acute and chronic inflammation, autoimmunity, and infection published in English after 2000, conducted in humans using the fluorescence method of quantifying DNA. Of the 442 articles retrieved, 83 were identified for full-text review and 13 remained after application of inclusion criteria. Of the reviewed studies, three involved acute inflammation, six involved chronic inflammation, and four involved infection. Healthy controls with interpretable results were included in six studies, three of which used the Quant-iT high-sensitivity DNA kit and found cfDNA quantities near 800 ng/ml, while the other three used other fluorescence methods and found quantities below 100 ng/ml. All 13 studies compared groups, and all but 1 found statistically significant differences between them. Among studies using the Quant-iT reagent, levels were higher in infection than in chronic inflammation. Among studies that used other reagents, levels increased from chronic to acute inflammation to severe infection. CfDNA levels were associated with mortality and with clinical outcomes in acute inflammation and infection. Most studies assessed cfDNA's correlation with other inflammation biomarkers and found inconclusive results. There appears to be an association between inflammation and cfDNA. Further research is necessary before cfDNA can be used clinically as a measure of inflammation. © The Author(s) 2016.

  18. DNA repair of cancer stem cells

    National Research Council Canada - National Science Library

    Mathews, Lesley A; Cabarcas, Stephanie M; Hurt, Elaine M

    2013-01-01

    ... leukemia by John E. Dick from the University of Toronto. The heterogeneity of human leukemia and the presence of stem cells in cancer was further translated into solid tumors by Al-Hajj et al. when they published a provocative paper in Proceedings of the National Academy of Sciences discussing the ability to distinguish tumorigenic (tumor-initi...

  19. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  20. 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.

  1. 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

  2. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and ...

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

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis...

  4. Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells.

    NARCIS (Netherlands)

    Vermeulen, C.; Bertocci, B.; Begg, A.C.; Vens, C.

    2007-01-01

    Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement

  5. Damage-induced DNA repair processes in Escherichia coli cells

    International Nuclear Information System (INIS)

    Slezarikova, V.

    1986-01-01

    The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

  6. DNA fingerprinting of the NCI-60 cell line panel.

    Science.gov (United States)

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  7. Fidelity of DNA Replication in Normal and Malignant Human Breast Cells.

    Science.gov (United States)

    1997-08-01

    polymerase. Biochemical and Biophysical Research Communications 21:6, 1965. 3.Farber, E. and Rubin, H. Cellular adaptation in the origin and development of...for DNA polymerase ca and in vitro SV40 DNA Cell culture. Suspension cultures of MDA MB-468 replication activities. human breast cells were adapted ...A vatit"Y Of DNA synthesis and the typt of DNA replica~tion Products " celular prca including DNA rsplicatlon. DNA repsair. R~NA formed in experiments

  8. DNA methylation and sensitivity to antimetabolites in cancer cell lines.

    Science.gov (United States)

    Sasaki, Shin; Kobunai, Takashi; Kitayama, Joji; Nagawa, Hirokazu

    2008-02-01

    The prediction of the cellular direction of metabolic pathways toward either DNA synthesis or DNA methylation is crucial for determining the susceptibility of cancers to anti-metabolites such as fluorouracil (5-FU). We genotyped the methylenetetrahydrofolate reductase (MTHFR) gene in NCI-60 cancer cell lines, and identified the methylation status of 24 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification. The susceptibility of the cancer cell lines to seven antimetabolites was then determined. Cells homozygous for CC at MTHFR-A1298C were significantly more sensitive to cyclocytidine, cytarabine (AraC) and floxuridine than those with AA or AC (p=0.0215, p=0.0166, and p=0.0323, respectively), and carried more methylated tumor suppressor genes (p=0.0313). Among the 12 tumor suppressor genes which were methylated in >25% of cancer cell lines, the methylation status of TIMP3, APC and IGSF4 significantly correlated with sensitivity to pyrimidine synthesis inhibitors. In particular, cells with methylated TIMP3 had reduced mRNA levels and were significantly more sensitive to aphidicolin-glycinate, AraC and 5-FU than cells with unmethylated TIMP3. We speculate that MTHFR-A1298C homozygous CC might direct the methylation rather than the synthesis of DNA, and result in the methylation of several tumor suppressor genes such as TIMP3. These genes could be useful biological markers for predicting the efficacy of antimetabolites.

  9. 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.

  10. DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells.

    Science.gov (United States)

    Tretyakova, Natalia Y; Michaelson-Richie, Erin D; Gherezghiher, Teshome B; Kurtz, Jamie; Ming, Xun; Wickramaratne, Susith; Campion, Melissa; Kanugula, Sreenivas; Pegg, Anthony E; Campbell, Colin

    2013-05-07

    Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

  11. DNA repair in mammalian nerve cells. 1. DNA synthesis in cerebral cortex induced by γ-irradiation of rats

    International Nuclear Information System (INIS)

    Ivanov, V.A.; Terpilovskaya, O.N.; Kulikov, A.V.; Tret'yak, T.M.

    1987-01-01

    A study was made of the DNA synthesis in cerebral cortex of rats, aged 14 and 60 days, after gamma-irradiation in vivo in a dose of 7 Gy, the 3 H-thymidine incorporation into DNA being determined. 137 Cs-radiation induces additional DNA synthesis in the neocortex tissue and in neurons. In the cortex of 14 day-old rats, the induced DNA synthesis stops 2 hours after irradiation, whereas in the cortex of 60 day-old rats and in neurons of rats of both the age groups DNA synthesis is proceeding for 3-35 hours. Specificity of DNA reparation, processes in nondividing cells is discussed

  12. Fidelity of a human cell DNA replication complex

    International Nuclear Information System (INIS)

    Roberts, J.D.; Kunkel, T.A.

    1988-01-01

    The authors have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZα-complementation gene, which encodes the α region (specifying the amino-terminal portion) of β-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase α-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes

  13. Fidelity of a human cell DNA replication complex

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, J.D.; Kunkel, T.A. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA))

    1988-10-01

    The authors have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ{alpha}-complementation gene, which encodes the {alpha} region (specifying the amino-terminal portion) of {beta}-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase {alpha}-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes.

  14. Rapid DNA replication origin licensing protects stem cell pluripotency.

    Science.gov (United States)

    Matson, Jacob Peter; Dumitru, Raluca; Coryell, Philip; Baxley, Ryan M; Chen, Weili; Twaroski, Kirk; Webber, Beau R; Tolar, Jakub; Bielinsky, Anja-Katrin; Purvis, Jeremy E; Cook, Jeanette Gowen

    2017-11-17

    Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-01-18

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

  16. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    state, may be of significant benefit for cfDNA research. Methods CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose...... environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.......Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...

  17. Nicotinamide; antioxidative and DNA hypomethylation effects in plant cells.

    Science.gov (United States)

    Berglund, Torkel; Wallström, Anders; Nguyen, Tuong-Van; Laurell, Cecilia; Ohlsson, Anna B

    2017-09-01

    The effects of nicotinamide (NIC) and its natural plant metabolites nicotinic acid (NIA) and trigonelline (TRIG) were studied with respect to defense in plant cell cultures. NIC and NIA could protect against oxidative stress damage caused by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which generates free radicals. Damage was analyzed as DNA strand breaks in cell cultures of Pisum sativum (garden pea), Daucus carota (carrot), Populus tremula L. × P. tremuloides (hybrid aspen) and Catharanthus roseus (Madagascar periwinkle), monitored by single cell gel electrophoresis (comet assay), and assays of cell leakage in C. roseus. The activities of aconitase and fumarase enzymes, which have key roles in energy metabolism, were analyzed in P. sativum cultures after treatment with NIC or NIA. Aconitase activity was increased by NIA, and fumarase activity was increased by both compounds. These compounds were shown to promote glutathione metabolism in P. sativum cultures, and NIC was shown to have a global DNA hypomethylating effect. Neither TRIG nor poly(ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide offered any protection against DNA damage or cell leakage, nor did they promote aconitase or fumarase activities, or glutathione metabolism. By this broad approach addressing multiple biochemical factors and different plant species, we demonstrate that NIC and NIA protect plant cells from oxidative stress, and that NIC clearly exerts an epigenetic effect; decreased DNA methylation. This indicates that these compounds have important roles in the regulation of metabolism in plant cells, especially in connection to stress. Copyright © 2017 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  18. Effects of physical, chemical or biological factors on DNA replication in mammalian cells. Part 2. Recovery of DNA complex, nucleoid and DNA replication after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Synzynys, B.I.; Kiseleva, V.I.; Trofimova, S.F.

    1984-11-01

    Murine LL cell line was employed in studies on the effects of gamma irradiation (6 Gy, 8 Gy/min from Co-60 source) on the kinetics and repair of DNA superstructure and recovery of DNA synthesis. Comparison of the kinetic data for the postradiation repair of the DNA-membrane complex, nucleoid, and recovery of DNA synthesis demonstrated that functional recovery of the former two factors preceded DNA synthesis by at least 2 h. In view of this lag, it appears that additional factors are involved in radiation damage that must be repaired before DNA synthesis commences. 10 references, 3 figures.

  19. Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

    Science.gov (United States)

    Zimny, Philip; Juncker, David; Reisner, Walter

    Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

  20. 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

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

    International Nuclear Information System (INIS)

    Kosaka, T.; Kuwabara, M.; Koide, F.

    1992-01-01

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

  2. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.

    Science.gov (United States)

    Bru, Samuel; Samper-Martín, Bàrbara; Quandt, Eva; Hernández-Ortega, Sara; Martínez-Laínez, Joan M; Garí, Eloi; Rafel, Marta; Torres-Torronteras, Javier; Martí, Ramón; Ribeiro, Mariana P C; Jiménez, Javier; Clotet, Josep

    2017-09-01

    Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. DNA computation in mammalian cells: microRNA logic operations.

    Science.gov (United States)

    Hemphill, James; Deiters, Alexander

    2013-07-17

    DNA computation can utilize logic gates as modules to create molecular computers with biological inputs. Modular circuits that recognize nucleic acid inputs through strand hybridization activate computation cascades to produce controlled outputs. This allows for the construction of synthetic circuits that can be interfaced with cellular environments. We have engineered oligonucleotide AND gates to respond to specific microRNA (miRNA) inputs in live mammalian cells. Both single and dual-sensing miRNA-based computation devices were synthesized for the cell-specific identification of endogenous miR-21 and miR-122. A logic gate response was observed with miRNA expression regulators, exhibiting molecular recognition of miRNA profile changes. Nucleic acid logic gates that are functional in a cellular environment and recognize endogenous inputs significantly expand the potential of DNA computation to monitor, image, and respond to cell-specific markers.

  4. 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.

  5. 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

  6. Mass spectrometry investigation of DNA adduct formation from bisphenol A quinone metabolite and MCF-7 cell DNA.

    Science.gov (United States)

    Zhao, Hongzhi; Wei, Juntong; Xiang, Li; Cai, Zongwei

    2018-05-15

    Bisphenol A (BPA) is a widely used additive in the plastic industry and has been reported to have genotoxicity. A hypothesis that BPA may enhance breast cancer risk through the formation of its metabolic intermediate or DNA adduct has been proposed. In this study, breast cancer cell MCF-7 was cultured and the cellular DNA was extracted from the cells. The adducts of bisphenol A 3,4-quinone (BPAQ) with 2'-deoxyguanosine (dG), calf thymus DNA and MCF-7 cell DNA were investigated. DNA adducts were characterized by using electrospray ionization Orbitrap high-resolution mass spectrometry and tandem mass spectrometry. The BPA-DNA adducts of BPAQ with dG, calf thymus and MCF-7 cell DNA were identified as 3-hydroxy-bisphenol A-N7-guanine (3-OH-BPA-N7Gua). The MS/MS fragmentation pathway of 3-OH-BPA-N7Gua was proposed based on obtained accurate mass data. BPA quinone metabolites can react with MCF-7 cell DNA in vitro. The findings provide evidence that BPA might covalently bind to DNA in MCF-7 cells mediated by quinone metabolites, which may increase our understanding of health risk associated with BPA exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors.

    Science.gov (United States)

    Pessôa, Rodrigo; Loureiro, Paula; Esther Lopes, Maria; Carneiro-Proietti, Anna B F; Sabino, Ester C; Busch, Michael P; Sanabani, Sabri S

    2016-01-01

    Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70). A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1). Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and

  8. 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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. 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. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  10. DNA-radiosensitivity and repair in mammolian cells

    International Nuclear Information System (INIS)

    Proskuryakov, S.Ya.; Ivannik, B.P.; Ryabchenko, N.I.

    1979-01-01

    Determination was made of the formation and repair of single-stranded DNA breaks (SB) in cells of rat thymus and liver and Ehrlich's ascites tumor (EAT) with the use of the method of low-gradient viscosimetry of alkaline cell lysates. The radiochemical yield of single-stranded breaks (Gsub(SB)) induced by irradiation of animals is 41.2 eV/break for hepatocytes, 96.8 eV/break, for thymocytes, and 129.7 eV/break, for EAT cells. The half-recovery time of single-stranded DNA breaks for cells of thymus and EAT exposed in vivo is 16.0 and 5.1 s -1 , correspondingly. In hepatocytes exposed in vivo and in vitro no repairs occurs for 3 h. Under conditions of inhibition of SB repair, when suspensions of thymocytes and hepatocytes were exposed in vitro at 4 deg C, Gsub(SB) is 35.5 and 38.7 eV/break, respectively. The analysis of the data obtained prompts the conclusion that under in vivo conditions, there is a correlation between DNA radiosensitivity and the rate of repair processes

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

    Energy Technology Data Exchange (ETDEWEB)

    Vitvitskii, V.N.

    1986-04-10

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

  12. Human papillomavirus type 16 DNA in periungual squamous cell carcinomas

    Energy Technology Data Exchange (ETDEWEB)

    Moy, R.L.; Eliezri, Y.D.; Bennett, R.G. (UCLA School of Medicine, Los Angeles, CA (USA)); Nuovo, G.J.; Siverstein, S. (UCLA School of Medicine, Los Angeles, CA (USA) Columbia Univ., New York, NY (USA)); Zitelli, J.A. (Montefiore Hospital, Pittsburgh, PA (USA))

    1989-05-12

    Ten squamous cell carcinomas (in situ or invasive) of the fingernail region were analyzed for the presence of DNA sequences homologous to human papilloma-virus (HPV) by dot blot hybridization. In most patients, the lesions were verrucae of long-term duration that were refractory to conventional treatment methods. Eight of the lesions contained HPV DNA sequences, and in six of these the sequences were related to HPV 16 as deduced from low-stringency nucleic acid hybridization followed by low- and high-stringency washes. Furthermore, the restriction endonuclease digestion pattern of DNA isolated from four of these lesions was diagnostic of episomal HPV 16. The high-frequency association of HPV 16 with periungual squamous cell carcinoma is similar to that reported for HPV 16 with squamous cell carcinomas on mucous membranes at other sites, notably the genital tract. The findings suggest that HPV 16 may play an important role in the development of squamous cell carcinomas of the finger, most notably those lesions that are chronic and located in the periungual area.

  13. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  14. 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

  15. Pulmonary endothelial cell DNA methylation signature in pulmonary arterial hypertension.

    Science.gov (United States)

    Hautefort, Aurélie; Chesné, Julie; Preussner, Jens; Pullamsetti, Soni S; Tost, Jorg; Looso, Mario; Antigny, Fabrice; Girerd, Barbara; Riou, Marianne; Eddahibi, Saadia; Deleuze, Jean-François; Seeger, Werner; Fadel, Elie; Simonneau, Gerald; Montani, David; Humbert, Marc; Perros, Frédéric

    2017-08-08

    Pulmonary arterial hypertension (PAH) is a severe and incurable pulmonary vascular disease. One of the primary origins of PAH is pulmonary endothelial dysfunction leading to vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Our objective was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. DNA was extracted from cultured PEC from idiopathic PAH ( n = 11), heritable PAH ( n = 10) and controls ( n = 18). DNA methylation was assessed using the Illumina HumanMethylation450 Assay. After normalization, samples and probes were clustered according to their methylation profile. Differential clusters were functionally analyzed using bioinformatics tools. Unsupervised hierarchical clustering allowed the identification of two clusters of probes that discriminates controls and PAH patients. Among 147 differential methylated promoters, 46 promoters coding for proteins or miRNAs were related to lipid metabolism. Top 10 up and down-regulated genes were involved in lipid transport including ABCA1, ABCB4, ADIPOQ, miR-26A, BCL2L11. NextBio meta-analysis suggested a contribution of ABCA1 in PAH. We confirmed ABCA1 mRNA and protein downregulation specifically in PAH PEC by qPCR and immunohistochemistry and made the proof-of-concept in an experimental model of the disease that its targeting may offer novel therapeutic options. In conclusion, DNA methylation analysis identifies a set of genes mainly involved in lipid transport pathway which could be relevant to PAH pathophysiology.

  16. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  17. 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 ...

  18. 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 ...

  19. 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 ...

  20. 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 ...

  1. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-04-15

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

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

    International Nuclear Information System (INIS)

    Braby, L.A.; Morgan, T.L.

    1992-01-01

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

  3. Molecular dosimetry of chemical mutagens: measurement of molecular dose and DNA repair germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1975-01-01

    Molecular dosimetry in the germ cells of male mice is reviewed with regard to in vivo alkylation of sperm heads, in vivo alkylation of sperm DNA, and possible alkylation of sperm protamine. DNA repair in male germ cells is reviewed with regard to basic design of experiments, DNA repair in various stages of spermatogenesis, effect of protamine on DNA repair following treatment with EMS or x radiation, and induction of DNA repair by methyl methanesulfonate, propyl methanesulfonate, and isopropyl methanesulfonate

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

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

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

  5. 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.

  6. 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.

  7. DNA hybrids suggesting a recombination process repairing radiation-induced DNA double-strand breaks in Ehrlich Ascites tumor cells

    International Nuclear Information System (INIS)

    Barthel, H.R.

    1984-01-01

    The results presented suggest the possibility of repair of DNA double-strand breaks by recombination, at least in the S and G 2 -phases of the cell cycle, in mammalian cells. Further experiments with synchronized cell cultures will have to show whether this process may also occur in the G 1 -phase of the cell cycle. (orig./AJ) [de

  8. Host DNA damage response factors localize to merkel cell polyomavirus DNA replication sites to support efficient viral DNA replication.

    Science.gov (United States)

    Tsang, Sabrina H; Wang, Xin; Li, Jing; Buck, Christopher B; You, Jianxin

    2014-03-01

    Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR-mediated DDR

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

    International Nuclear Information System (INIS)

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

    2005-01-01

    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 [ 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 [ 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

  10. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  11. DNA methylation supports intrinsic epigenetic memory in mammalian cells.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

  12. 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,

  13. The impact of impaired DNA damage responses on cells, tissues and organisms

    NARCIS (Netherlands)

    Yi, Xia

    2007-01-01

    Current cancer therapies rely mainly on DNA damaging insults (irradiation, DNA alkylating agents, DNA synthesis inhibitors etc.). The rationale behind these treatments is that rapidly growing cancer cells suffer more from DNA damaging insults. Unfortunately, the majority of current therapies fail to

  14. Sedimentation properties of DNA-membrane complexes and yield of DNA breaks at irradiation of mammalian cells

    International Nuclear Information System (INIS)

    Erzgraber, G.; Kozubek, S.; Lapidus, I.L.

    1985-01-01

    The dependence of the relative sedimentation velocity of DNA-membrane complexes on the dose of irradiation and time of incubation of Chinese Hamster cells is analysed. It is concluded that the initial part of the curve provides the information on the occurrence of single strand breaks in DNA; the position of the local maximum allows us to calculate the yield of DNA double strand breaks. The reparation decay constant can be estimated as well

  15. DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

    Science.gov (United States)

    Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

    2012-03-01

    Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells

    DEFF Research Database (Denmark)

    Kirkin, Alexei F.; Dzhandzhugazyan, Karine N.; Guldberg, Per

    2018-01-01

    In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT...... antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25...... patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can...

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

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott

    2014-01-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial...... slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I...... 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...

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  19. Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2[W

    Science.gov (United States)

    Amoroso, Alessandra; Concia, Lorenzo; Maggio, Caterina; Raynaud, Cécile; Bergounioux, Catherine; Crespan, Emmanuele; Cella, Rino; Maga, Giovanni

    2011-01-01

    The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes. PMID:21325140

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

    International Nuclear Information System (INIS)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-01-01

    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

  1. 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

  2. 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.

  3. File list: Oth.Unc.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.20.DNA-RNA_hybrids.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.05.DNA-RNA_hybrids.AllCell.bed ...

  6. File list: Oth.Unc.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.10.DNA-RNA_hybrids.AllCell.bed ...

  7. File list: Oth.YSt.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.50.DNA-RNA_hybrids.AllCell.bed ...

  8. File list: Oth.Unc.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Unclassif...ied http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.05.DNA-RNA_hybrids.AllCell.bed ...

  9. File list: Oth.YSt.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.20.DNA-RNA_hybrids.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.10.DNA-RNA_hybrids.AllCell.bed ...

  11. 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, Bente Klarlund

    2005-01-01

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

  12. 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. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

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

    Energy Technology Data Exchange (ETDEWEB)

    Brozmanova, J.; Masek, F.; Synzynys, B.I.; Saenko, A.S.

    1985-11-05

    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).

  14. DNA replication in Chinese hamster ovary cells made permeable to nucleotides by Tween-80 treatment

    Energy Technology Data Exchange (ETDEWEB)

    Billen, D.; Olson, A.C.

    1976-01-01

    We have developed a method for permeabilizing CHO cells to nucleotides under conditions which allow most cells to remain viable. Permeabilized cells can carry out ATP-dependent, semiconservative synthesis of DNA. The data are consistent with the continuation of DNA synthesis in those cells in S phase at the time of treatment, possibly limited to completion of replicon synthesis without new initiations.

  15. [Methylcobalamin-dependent enzymatic methylation of DNA in a cell-free extract of Propionibacterium shermanii].

    Science.gov (United States)

    Antoshkina, N V; Vorob'eva, L I; Bur'ianov, Ia I

    1981-01-01

    The regulation of the functional activity of Propionibacterium shermanii cells by cobalamin is accompanied by an increase in the methylation of their DNA at the cytosine residue: the DNA from B12-deficient cells is undermethylated as compared with the DNA from control cells, i. e. cells synthesizing corrinoids. The results of in vitro experiments make it possible to correlate this phenomenon with the capability of DNA methylases from B12-deficient and control cells to function with different donors of methyl groups. Just as the enzymes methylating adenine and cytosine in B12-deficient cells, adenine DNA methylase from cells synthesizing corrinoids is active in vitro with S-adenosylmethionine. Cytosine DNA methylase from P. shermanii control cells is inactive with S-adenosylmethionine and transfers methyl groups only in the presence of cobalamins.

  16. Evaluation of HTLV-1 activity in HAM/TSP patients using proviral load and Tax mRNA expression after In Vitro lymphocyte activation

    Directory of Open Access Journals (Sweden)

    Atefeh Yari

    2014-07-01

    Conclusion: Although proviral load had been addressed as a valuable index for monitoring HTLV-1 infected subjects, the results of this study demonstrated that Tax expression in activated PBMCs along with proviral load assessment in HAM/TSP patients are a more reliable factor for determining the prognosis and monitoring healthy carriers and HAM/TSP patients.

  17. Repair of ionizing radiation damage in primate αDNA transfected into rat cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.

    1992-01-01

    The time-course of repair of irradiated primate αDNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no αDNA. Plasmid pBUC4α10, containing 10 tandem 172 bp αDNA subunits in its 5kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected αDNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected αDNA in time-course experiments. αDNA continuously entered nuclei for processing in the first 2.5h after transfection. The pool of damaged bases in αDNA in NRK nuclei was detectable 2.5 h after transfection. (author)

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

    International Nuclear Information System (INIS)

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H.

    1991-01-01

    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

  19. DNA methylation in states of cell physiology and pathology.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2007-10-01

    Full Text Available DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation. The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences five new genes which are potential biomarkers of lung cancer have been presented.

  20. Association of Sicca Syndrome with Proviral Load and Proinflammatory Cytokines in HTLV-1 Infection

    Directory of Open Access Journals (Sweden)

    Clara Mônica Lima

    2016-01-01

    Full Text Available The Sjögren syndrome has been diagnosed in patients with HTLV-1 associated myelopathy and dry mouth and dry eyes are documented in HTLV-1 carriers. However the diagnosis of Sjögren syndrome in these subjects has been contested. In this cross-sectional study, we evaluated the role of immunological factors and proviral load, in sicca syndrome associated with HTLV-1 in patients without myelopathy. Subjects were recruited in the HTLV-1 Clinic, from 2009 to 2011. The proviral load and cytokine levels (IFN-γ, TNF-α, IL-5, and IL-10 were obtained from a database containing the values presented by the subjects at admission in the clinic. Of the 272 participants, 59 (21.7% had sicca syndrome and in all of them anti-Sjögren syndrome related antigen A (SSA and antigen B (SSB were negatives. The production of TNF-α and IFN-γ was higher in the group with sicca syndrome (P<0.05 than in HTLV-1 infected subjects without sicca syndrome. Our data indicates that patients with sicca syndrome associated with HTLV-1 do not have Sjögren syndrome. However the increased production of TNF-α and IFN-γ in this group of patients may contribute to the pathogenesis of sicca syndrome associated with HTLV-1.

  1. Mitochondrial DNA-depleted A549 cells are resistant to bleomycin

    Science.gov (United States)

    Brar, Sukhdev S.; Meyer, Joel N.; Bortner, Carl D.; Van Houten, Bennett

    2012-01-01

    Alveolar epithelial cells are considered to be the primary target of bleomycin-induced lung injury, leading to interstitial fibrosis. The molecular mechanisms by which bleomycin causes this damage are poorly understood but are suspected to involve generation of reactive oxygen species and DNA damage. We studied the effect of bleomycin on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in human alveolar epithelial A549 cells. Bleomycin caused an increase in reactive oxygen species production, DNA damage, and apoptosis in A549 cells; however, bleomycin induced more mtDNA than nDNA damage. DNA damage was associated with activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and cleavage and activation of protein kinase D1 (PKD1), a newly identified mitochondrial oxidative stress sensor. These effects appear to be mtDNA-dependent, because no caspase-3 or PKD1 activation was observed in mtDNA-depleted (ρ0) A549 cells. Survival rate after bleomycin treatment was higher for A549 ρ0 than A549 cells. These results suggest that A549 ρ0 cells are more resistant to bleomycin toxicity than are parent A549 cells, likely in part due to the depletion of mtDNA and impairment of mitochondria-dependent apoptotic pathways. PMID:22773697

  2. 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.

  3. DNA Measurement of Overlapping Cell Nuclei in Thick Tissue Sections

    Directory of Open Access Journals (Sweden)

    Liang Ji

    1997-01-01

    Full Text Available The paper describes an improved image analysis procedure for measuring the DNA content of cell nuclei in thick sections of liver tissue by absorption densitometry. Whereas previous methods only permitted the analysis of isolated nuclei, the new technique enables both isolated and overlapping nuclei to be measured. A 3D segmentation procedure determines whether each object is an isolated nucleus or a pair of overlapping nuclei; in the latter case the combined optical density is redistributed to the individual nuclei. A selection procedure ensures that only complete nuclei are measured.

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

    Science.gov (United States)

    Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

    2016-01-01

    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.

  5. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

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

  7. 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.

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

    Science.gov (United States)

    Song, Eun Ah; Kim, Hyeyoung

    2016-08-03

    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.

  9. 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.

  10. In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

    International Nuclear Information System (INIS)

    Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

    1988-01-01

    Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

  11. In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

    Energy Technology Data Exchange (ETDEWEB)

    Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M. (Washington Univ., St. Louis, MO (USA))

    1988-09-20

    Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K{sub m} values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 {mu}M. For UV-induced DNA repair synthesis, the apparent K{sub m} values were substantially lower, ranging from 0.11 to 0.44 {mu}M for AG1518 cells and from 0.06 to 0.24 {mu}M for IMR-90 cells. Recent data implicate DNA polymerase {delta} in UV-induced repair synthesis and suggest that DNA polymerases {alpha} and {delta} are both involved in semiconservative replication. They measured K{sub m} values for dGTP and dTTP for polymerases {alpha} and {delta}, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K{sub m} values for DNA polymerase {delta} are much greater than the K{sub m} values for UV-induced repair synthesis, suggesting that when polymerase {delta} functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K{sub m} values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K{sub m} for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo.

  12. RUNX1 regulates site specificity of DNA demethylation by recruitment of DNA demethylation machineries in hematopoietic cells.

    Science.gov (United States)

    Suzuki, Takahiro; Shimizu, Yuri; Furuhata, Erina; Maeda, Shiori; Kishima, Mami; Nishimura, Hajime; Enomoto, Saaya; Hayashizaki, Yoshihide; Suzuki, Harukazu

    2017-09-12

    RUNX1 is an essential master transcription factor in hematopoietic development and plays important roles in immune functions. Although the gene regulatory mechanism of RUNX1 has been characterized extensively, the epigenetic role of RUNX1 remains unclear. Here, we demonstrate that RUNX1 contributes DNA demethylation in a binding site-directed manner in human hematopoietic cells. Overexpression analysis of RUNX1 showed the RUNX1-binding site-directed DNA demethylation. The RUNX1-mediated DNA demethylation was also observed in DNA replication-arrested cells, suggesting an involvement of active demethylation mechanism. Coimmunoprecipitation in hematopoietic cells showed physical interactions between RUNX1 and DNA demethylation machinery enzymes TET2, TET3, TDG, and GADD45. Further chromatin immunoprecipitation sequencing revealed colocalization of RUNX1 and TET2 in the same genomic regions, indicating recruitment of DNA demethylation machinery by RUNX1. Finally, methylome analysis revealed significant overrepresentation of RUNX1-binding sites at demethylated regions during hematopoietic development. Collectively, the present data provide evidence that RUNX1 contributes site specificity of DNA demethylation by recruitment of TET and other demethylation-related enzymes to its binding sites in hematopoietic cells.

  13. Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens.

    Science.gov (United States)

    Shoura, Massa J; Gabdank, Idan; Hansen, Loren; Merker, Jason; Gotlib, Jason; Levene, Stephen D; Fire, Andrew Z

    2017-10-05

    Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples. Copyright © 2017 Shoura et al.

  14. Early antiretroviral therapy reduces HIV DNA following perinatal HIV infection.

    Science.gov (United States)

    Foster, Caroline; Pace, Matthew; Kaye, Steve; Hopkins, Emily; Jones, Mathew; Robinson, Nicola; Mant, Christine; Cason, John; Fidler, Sarah; Frater, John

    2017-08-24

    : The impact of antiretroviral therapy (ART) on the size of the HIV reservoir has implications for virological remission in adults, but is not well characterized in perinatally acquired infection. In a prospective observational study of 20 children with perinatally acquired infection and sustained viral suppression on ART for more than 5 years, proviral DNA was significantly higher in deferred (>4 years) versus early (first year of life) ART recipients (P = 0.0062), and correlated with age of initiation (P = 0.13; r = 0.57). No difference was seen in cell-associated viral RNA (P = 0.36). Identifying paediatric populations with smaller reservoirs may inform strategies with potential to induce ART-free remission.

  15. Identification of structural DNA variations in human cell cultures after long-term passage.

    Science.gov (United States)

    Pavlova, G V; Vergun, A A; Rybalkina, E Y; Butovskaya, P R; Ryskov, A P

    2015-01-01

    Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8-12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.

  16. 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.

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

    International Nuclear Information System (INIS)

    Olive, P.L.; Hilton, J.; Durand, R.E.

    1986-01-01

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

  18. 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.

  19. The FAS-670 AA genotype is associated with high proviral load in peruvian HAM/TSP patients.

    Science.gov (United States)

    Rosado, Jason; Morales, Sandra; López, Giovanni; Clark, Daniel; Verdonck, Kristien; Gotuzzo, Eduardo; Van Camp, Guy; Talledo, Michael

    2017-04-01

    Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Apoptosis is a mechanism of defense elicited by many triggers, including cross-linking of the FAS receptor expressed in viruses-infected cells, and the ligand FASL presented by T-cytotoxic cells. As HAM/TSP has been associated with high levels of proviral load (PVL), we hypothesized that certain genotypes of single-nucleotide polymorphisms (SNPs) associated with a decreased protein expression of FAS and FASL could be risk factors for this disease. Three SNPs: FAS-670A/G (rs1800682), FAS-1377G/A (rs2234767), and FASL-844C/T (rs763110) were analyzed in 73 HAM/TSP patients and 143 HTLV-1 asymptomatic carriers. Ancestry informative markers were used to adjust for ethnicity through a principal component analysis. Gender, age, PVL, and the first three principal components were used as covariates. The FAS/FASL genotype distribution was not associated with HAM/TSP presence (P-> 0.05). The FAS-670 AA genotype was associated with high PVL in comparison to FAS-670 GG in HAM/TSP patients (P = 0.015), while in asymptomatic carriers low levels of PVL were observed (P > 0.05). Our findings suggest that rs1800682, rs2234767, and rs763110 genotypes are not associated with the presence of HAM/TSP, but that the FAS-670 AA genotype can promote higher PVL values in HAM/TSP patients. J. Med. Virol. 89:726-731, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    Science.gov (United States)

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  1. 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.

  2. 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....

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

    Directory of Open Access Journals (Sweden)

    Yingrui Li

    2010-11-01

    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.

  4. DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

    Science.gov (United States)

    Sokol, Anna M.; Cruet-Hennequart, Séverine; Pasero, Philippe; Carty, Michael P.

    2013-11-01

    Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

  5. OCT4: dynamic DNA binding pioneers stem cell pluripotency.

    Science.gov (United States)

    Jerabek, Stepan; Merino, Felipe; Schöler, Hans Robert; Cojocaru, Vlad

    2014-03-01

    OCT4 was discovered more than two decades ago as a transcription factor specific to early embryonic development. Early studies with OCT4 were descriptive and looked at determining the functional roles of OCT4 in the embryo as well as in pluripotent cell lines derived from embryos. Later studies showed that OCT4 was one of the transcription factors in the four-factor cocktail required for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and that it is the only factor that cannot be substituted in this process by other members of the same protein family. In recent years, OCT4 has emerged as a master regulator of the induction and maintenance of cellular pluripotency, with crucial roles in the early stages of differentiation. Currently, mechanistic studies look at elucidating the molecular details of how OCT4 contributes to establishing selective gene expression programs that define different developmental stages of pluripotent cells. OCT4 belongs to the POU family of proteins, which have two conserved DNA-binding domains connected by a variable linker region. The functions of OCT4 depend on its ability to recognize and bind to DNA regulatory regions alone or in cooperation with other transcription factors and on its capacity to recruit other factors required to regulate the expression of specific sets of genes. Undoubtedly, future iPSC-based applications in regenerative medicine will benefit from understanding how OCT4 functions. Here we provide an integrated view of OCT4 research conducted to date by reviewing the different functional roles for OCT4 and discussing the current progress in understanding their underlying molecular mechanisms. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. © 2013.

  6. An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

    Science.gov (United States)

    Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

    2017-12-19

    Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

  7. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation.

    Science.gov (United States)

    Strzalka, Wojciech; Ziemienowicz, Alicja

    2011-05-01

    PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

  8. 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

  9. Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA*

    Science.gov (United States)

    Zhou, Yayan; Hingorani, Manju M.

    2012-01-01

    Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43–54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O'Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen

  10. Targeting Ongoing DNA Damage in Multiple Myeloma: Effects of DNA Damage Response Inhibitors on Plasma Cell Survival

    Directory of Open Access Journals (Sweden)

    Ana Belén Herrero

    2017-05-01

    Full Text Available Human myeloma cell lines (HMCLs and a subset of myeloma patients with poor prognosis exhibit high levels of replication stress (RS, leading to DNA damage. In this study, we confirmed the presence of DNA double-strand breaks (DSBs in several HMCLs by measuring γH2AX and RAD51 foci and analyzed the effect of various inhibitors of the DNA damage response on MM cell survival. Inhibition of ataxia telangiectasia and Rad3-related protein (ATR, the main kinase mediating the response to RS, using the specific inhibitor VE-821 induced more cell death in HMCLs than in control lymphoblastoid cells and U266, an HMCL with a low level of DNA damage. The absence of ATR was partially compensated by ataxia telangiectasia-mutated protein (ATM, since chemical inhibition of both kinases using VE-821 and KU-55933 significantly increased the death of MM cells with DNA damage. We found that ATM and ATR are involved in DSB repair by homologous recombination (HR in MM. Inhibition of both kinases resulted in a stronger inhibition that may underlie cell death induction, since abolition of HR using two different inhibitors severely reduced survival of HMCLs that exhibit DNA damage. On the other hand, inhibition of the other route involved in DSB repair, non-homologous end joining (NHEJ, using the DNA-PK inhibitor NU7441, did not affect MM cell viability. Interestingly, we found that NHEJ inhibition did not increase cell death when HR was simultaneously inhibited with the RAD51 inhibitor B02, but it clearly increased the level of cell death when HR was inhibited with the MRE11 inhibitor mirin, which interferes with recombination before DNA resection takes place. Taken together, our results demonstrate for the first time that MM cells with ongoing DNA damage rely on an intact HR pathway, which thereby suggests therapeutic opportunities. We also show that inhibition of HR after the initial step of end resection might be more appropriate for inducing MM cell death, since it

  11. 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.

  12. Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

    Science.gov (United States)

    Wu, Min; Li, Jin; Yue, Lei; Bai, Lu; Li, Yaming; Chen, Jieliang; Zhang, Xiaonan; Yuan, Zhenghong

    2018-04-01

    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  13. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  14. Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

    Science.gov (United States)

    Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

    2015-01-01

    Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

  15. Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation

    Science.gov (United States)

    Goswami, Gunajit; Boruah, Himangshu; Gautom, Trishnamoni; Jyoti Hazarika, Dibya; Barooah, Madhumita; Boro, Robin Chandra

    2017-12-01

    Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

  16. DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis.

    Science.gov (United States)

    Kanemaru, Yuki; Suzuki, Tetsuya; Sassa, Akira; Matsumoto, Kyomu; Adachi, Noritaka; Honma, Masamitsu; Numazawa, Satoshi; Nohmi, Takehiko

    2017-01-01

    Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA. We introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay. These results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and

  17. 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.

  18. Covalently bound DNA on naked iron oxide nanoparticles: Intelligent colloidal nano-vector for cell transfection.

    Science.gov (United States)

    Magro, Massimiliano; Martinello, Tiziana; Bonaiuto, Emanuela; Gomiero, Chiara; Baratella, Davide; Zoppellaro, Giorgio; Cozza, Giorgio; Patruno, Marco; Zboril, Radek; Vianello, Fabio

    2017-11-01

    Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration.

    Directory of Open Access Journals (Sweden)

    Soumya Gupta

    Full Text Available Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.

  20. Telomeres in ICF syndrome cells are vulnerable to DNA damage due to elevated DNA:RNA hybrids

    Science.gov (United States)

    Sagie, Shira; Toubiana, Shir; Hartono, Stella R.; Katzir, Hagar; Tzur-Gilat, Aya; Havazelet, Shany; Francastel, Claire; Velasco, Guillaume; Chédin, Frédéric; Selig, Sara

    2017-01-01

    DNA:RNA hybrids, nucleic acid structures with diverse physiological functions, can disrupt genome integrity when dysregulated. Human telomeres were shown to form hybrids with the lncRNA TERRA, yet the formation and distribution of these hybrids among telomeres, their regulation and their cellular effects remain elusive. Here we predict and confirm in several human cell types that DNA:RNA hybrids form at many subtelomeric and telomeric regions. We demonstrate that ICF syndrome cells, which exhibit short telomeres and elevated TERRA levels, are enriched for hybrids at telomeric regions throughout the cell cycle. Telomeric hybrids are associated with high levels of DNA damage at chromosome ends in ICF cells, which are significantly reduced with overexpression of RNase H1. Our findings suggest that abnormally high TERRA levels in ICF syndrome lead to accumulation of telomeric hybrids that, in turn, can result in telomeric dysfunction. PMID:28117327

  1. Evaluation of the role of TAX, HBZ, and HTLV-1 proviral load on the survival of ATLL patients.

    Science.gov (United States)

    Akbarin, Mohammad Mehdi; Shirdel, Abbas; Bari, Alireza; Mohaddes, Seyedeh Tahereh; Rafatpanah, Houshang; Karimani, Ehsan Ghayour; Etminani, Kobra; Golabpour, Amin; Torshizi, Reza

    2017-06-01

    Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy with very poor prognosis and short survival, caused by the human T-lymphotropic virus type-1 (HTLV-1). The HTLV-1 biomarkers trans-activator x (TAX) and HTLV-1 basic leucine zipper factor (HBZ) are main oncogenes and life-threatening elements. This study aimed to assess the role of the TAX and HBZ genes and HTLV-1 proviral load (PVL) in the survival of patients with ATLL. Forty-three HTLV-1-infected individuals, including 18 asymptomatic carriers (AC) and 25 ATLL patients (ATLL), were evaluated between 2011 and 2015. The mRNA expression of TAX and HBZ and the HTLV-1 PVL were measured by quantitative PCR. Significant differences in the mean expression levels of TAX and HBZ were observed between the two study groups (ATLL and AC, P =0.014 and P =0.000, respectively). In addition, the ATLL group showed a significantly higher PVL than AC ( P =0.000). There was a significant negative relationship between PVL and survival among all study groups ( P =0.047). The HTLV-1 PVL and expression of TAX and HBZ were higher in the ATLL group than in the AC group. Moreover, a higher PVL was associated with shorter survival time among all ATLL subjects. Therefore, measurement of PVL, TAX , and HBZ may be beneficial for monitoring and predicting HTLV-1-infection outcomes, and PVL may be useful for prognosis assessment of ATLL patients. This research demonstrates the possible correlation between these virological markers and survival in ATLL patients.

  2. 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.

  3. DNA repair proteins in cells of the human immune system; Le proteine della riparazione del DNA in cellule del sistema immunitario umano

    Energy Technology Data Exchange (ETDEWEB)

    Frasca, D.; Barattini, P.; Guidi, F.; Scarpaci, S. [ENEA, Sez. Tossicologia e Scienze Biomediche, Rome (Italy); Doria, G. [Rome Univ. Tor Vergata, Rome (Italy). Cattedra di Immunologia

    2001-02-01

    Human longevity depends on the efficiency of DNA repair mechanisms. In irradiated cells of the human immune system, the principal repair mechanism involves the DNA-Pk protein complex. [Italian] La durata della vita dipende dalla efficienza di meccanismi di riparazione del DNA. Nelle cellule del sistema immunitario umano danneggiate il principale meccanismo di riparazione coinvolge il complesso proteico DNA-PK.

  4. DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

    Science.gov (United States)

    Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

    2012-01-01

    The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

  5. Cell-free placental DNA beyond Down syndrome: Lessons learned from fetal RHD genotyping

    NARCIS (Netherlands)

    Thurik, F.F.

    2016-01-01

    In this thesis research is presented on cell-free fetal DNA (cffDNA), which is present in plasma and serum of pregnant women. This fetal DNA can be used for fetal genotyping, but may also give indirect information on pregnancy and pregnancy outcome. The research consists of two sections. In the

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

    Science.gov (United States)

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

  7. 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

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

    International Nuclear Information System (INIS)

    Dizdaroglu, Miral

    1999-01-01

    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

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

    International Nuclear Information System (INIS)

    Povirk, L.F.

    1977-01-01

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

  10. Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

    Science.gov (United States)

    Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

    2017-11-10

    Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering

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

    International Nuclear Information System (INIS)

    Marie, Melanie; Hafner, Sophie; Moratille, Sandra; Vaigot, Pierre; Rigaud, Odile; Martin, Michele T.; Mine, Solene

    2012-01-01

    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)

  12. Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation.

    Science.gov (United States)

    Weeden, Clare E; Asselin-Labat, Marie-Liesse

    2018-01-01

    Maintenance of genomic integrity in tissue-specific stem cells is critical for tissue homeostasis and the prevention of deleterious diseases such as cancer. Stem cells are subject to DNA damage induced by endogenous replication mishaps or exposure to exogenous agents. The type of DNA lesion and the cell cycle stage will invoke different DNA repair mechanisms depending on the intrinsic DNA repair machinery of a cell. Inappropriate DNA repair in stem cells can lead to cell death, or to the formation and accumulation of genetic alterations that can be transmitted to daughter cells and so is linked to cancer formation. DNA mutational signatures that are associated with DNA repair deficiencies or exposure to carcinogenic agents have been described in cancer. Here we review the most recent findings on DNA repair pathways activated in epithelial tissue stem and progenitor cells and their implications for cancer mutational signatures. We discuss how deep knowledge of early molecular events leading to carcinogenesis provides insights into DNA repair mechanisms operating in tumours and how these could be exploited therapeutically. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  14. 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.

  15. Interaction of large DNA viruses with dendritic cells.

    Science.gov (United States)

    Jenne, L; Thumann, P; Steinkasserer, A

    2001-12-01

    Dendritic cells (DC) with their unique capacity to prime naïve T cells are crucial in the induction of immunological responses, including anti-tumoral and anti-viral immunity. DC based immunotherapies are thus currently considered a particularly promising approach for cellular immunotherapy. The cloning of tumor associated antigens (TAAs) together with the possibility of manipulating viral genomes by biotechnological techniques has sparked the interest of using genetically modified viruses to transduce DC in order to achieve antigenic expression of TAA with the aim of inducing a protective immune response. An increasing number of modified viral vectors has been designed for gene therapy purposes and consecutively has been used for the ex vivo transduction of DC. It has been shown that viral vectors genetically engineered to express TAA or immune modifiers like cytokines or costimulatory molecules can lead to a high level of transgene expression. Furthermore, these studies have also revealed that viruses have developed several immune evasion mechanisms specifically targeting DC. Therefore, analysing the interactions of viruses with DC is crucial for the development of new viral vectors suitable for the transduction of DC. In this report we describe the interaction of two large DNA viruses, herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV), with DC generated from peripheral blood mononuclear cells.

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

    International Nuclear Information System (INIS)

    Rakic, P.

    1985-01-01

    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 [ 3 H] thymidine at ages ranging from 6 postnatal months to 17 years

  17. Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability.

    Science.gov (United States)

    Brundin, M; Figdor, D; Sundqvist, G; Sjögren, U

    2015-01-01

    To investigate whether DNA from two obligate anaerobes, Fusobacterium nucleatum and Peptostreptococcus anaerobius, is recoverable after loss of cell viability induced by air exposure. Harvested cultures of F. nucleatum and P. anaerobius were killed by exposure to air and stored in phosphate-buffered saline. Dead cells were incubated aerobically for up to 6 months. Every month, the presence of detectable DNA in the cell pellet and supernatant was assessed by conventional and quantitative PCR. Cell staining techniques were used to characterize the cell wall permeability of air-killed cells. Scanning electron microscopy was used to examine viable, freshly killed and stored cells. With conventional PCR, amplifiable DNA was detectable over 6 months in all samples. Quantitative PCR showed a progressive fall in DNA concentration in nonviable cell pellets and a concomitant rise in DNA concentration in the supernatant. DNA staining showed that some air-killed cells retained an intact cell wall. After storage, SEM of both air-killed species revealed shrivelling of the cells, but some cells of P. anaerobius retained their initial form. Amplifiable DNA from F. nucleatum and P. anaerobius was detectable 6 months after loss of viability. Air-killed anaerobes initially retained their cell form, but cells gradually shriveled over time. The morphological changes were more pronounced with the gram-negative F. nucleatum than the gram-positive P. anaerobius. Over 6 months, there was a gradual increase in cell wall permeability with progressive leakage of DNA. Bacterial DNA was recoverable long after loss of cell viability. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  18. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

    DEFF Research Database (Denmark)

    Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris

    2016-01-01

    Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods...... for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA...... DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA...

  19. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.

    Science.gov (United States)

    Gong, J; Traganos, F; Darzynkiewicz, Z

    1994-05-01

    In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safety transported prior to analysis.

  20. 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.

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

    International Nuclear Information System (INIS)

    Yu, L.D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-01-01

    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

  2. Clinical applications of urinary cell-free DNA in cancer: current insights and promising future.

    Science.gov (United States)

    Lu, Tian; Li, Jinming

    2017-01-01

    Liquid biopsy is gaining significant attention as a tool for unveiling the molecular landscape of tumor and holds great promise for individualized medicine for cancer. Cell-free DNA serves as an extremely important component of liquid biopsy for cancer, and cell-free DNA in urine is even promising due to the remarkable advantage of urine as an ultra-noninvasive sample source over tissue and blood. Compared with the widely studied cell-free DNA in blood, less is known about the role of urinary cell-free DNA. Urinary cell-free DNA has the ability to give comprehensive and crucial information on cancer as it carries genetic messages from cells shedding directly into urine as well as transporting from circulation. As an indispensable component of liquid biopsy, urinary cell-free DNA is believed to have the potential of being a useful and ultra-noninvasive tool for cancer screening, diagnosis, prognosis, and monitoring of cancer progression and therapeutic effect. In this review, we provide the current insights into the clinical applications of urinary cell-free DNA in cancer. We also introduce the basic biological significance and some technical issues in the detection of urinary cell-free DNA.

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

    International Nuclear Information System (INIS)

    Lee, E.W.; Johnson, J.T.; Garner, C.D.

    1989-01-01

    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 [ 3 H]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 [ 3 H]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

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

    International Nuclear Information System (INIS)

    Kim, Sylvia Jeewon; Turner, Scott; Killion, Salena; Hellerstein, Marc K.

    2005-01-01

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

  5. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  6. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  7. Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

    OpenAIRE

    Robinson, H L; Zinkus, D M

    1990-01-01

    Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the ...

  8. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    Science.gov (United States)

    Kim, G. J.; Kim, W.; Kim, K. T.; Lee, J. K.

    2010-01-01

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  9. [Effect of cobalamin derivatives on DNA synthesis in cells of Propionibacterium freudenreichii subsp. shermanii].

    Science.gov (United States)

    Iordan, E P; Petukhova, N I; Vorob'eva, L I

    1987-01-01

    The increase of DNA-synthesis rate (according incorporation [8-14C]adenine) in B12-deficient cells Propionibacterium shermanii as a result of different cobalamines adding into the cell suspension including metoxyethyladenile analog of adenozilcobalamin and some components of vitamin B12 molecule has been found. The DNA-synthesis rate in B12-deficient cells is nearly twice lower as compared with one in B12-normal cells. Considerable stimulative effect (80-100%) was provided with coenzyme forms of cobalamin. The data confirm the participation of vitamin B12 in DNA-synthesis in Propionibacterium cells.

  10. Transcription is Associated with Z-DNA Formation in Metabolically Active Permeabilized Mammalian Cell Nuclei

    Science.gov (United States)

    Wittig, Burghardt; Dorbic, Tomislav; Rich, Alexander

    1991-03-01

    Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.

  11. DNA methylation and transcriptional trajectories during human development and reprogramming of isogenic pluripotent stem cells

    NARCIS (Netherlands)

    Roost, Matthias S; Slieker, Roderick C; Bialecka, Monika; van Iperen, Liesbeth; Gomes Fernandes, Maria M; He, Nannan; Suchiman, H Eka D; Szuhai, Karoly; Carlotti, Françoise; de Koning, Eelco J P; Mummery, Christine L; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2017-01-01

    Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal

  12. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available This presentation is about the photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses. It outlines the background on embryonic stem cells (ES) and phototransfection....

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

    International Nuclear Information System (INIS)

    Leffke, A.; Helle, N.; Boegl, K.W.; Schreiber, G.A.

    1993-01-01

    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.)

  14. DNA damage follows repair factor depletion and portends genome variation in cancer cells after pore migration

    OpenAIRE

    Irianto, Jerome; Xia, Yuntao; Pfeifer, Charlotte R.; Athirasala, Avathamsa; Ji, Jiazheng; Alvey, Cory; Tewari, Manu; Bennett, Rachel; Harding, Shane M.; Liu, Andrea; Greenberg, Roger A.; Discher, Dennis E.

    2016-01-01

    Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1 – a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSC) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic ‘comet’ measurements. Migration also causes multiple DNA repair proteins to segregate away from D...

  15. Metabolic rescue in pluripotent cells from patients with mtDNA disease.

    Science.gov (United States)

    Ma, Hong; Folmes, Clifford D L; Wu, Jun; Morey, Robert; Mora-Castilla, Sergio; Ocampo, Alejandro; Ma, Li; Poulton, Joanna; Wang, Xinjian; Ahmed, Riffat; Kang, Eunju; Lee, Yeonmi; Hayama, Tomonari; Li, Ying; Van Dyken, Crystal; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Koski, Amy; Mitalipov, Nargiz; Amato, Paula; Wolf, Don P; Huang, Taosheng; Terzic, Andre; Laurent, Louise C; Izpisua Belmonte, Juan Carlos; Mitalipov, Shoukhrat

    2015-08-13

    Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

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

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1994-01-01

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

  17. 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

  18. Radioadaptive response. Efficient repair of radiation-induced DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji; Aritomi, Hisako; Morisita, Jun

    1996-01-01

    To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage

  19. Murine scid cells complement ataxia-telangiectasia cells and show a normal port-irradiation response of DNA synthesis

    International Nuclear Information System (INIS)

    Komatsu, K.; Yoshida, M.; Okumura, Y.

    1993-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a deficit in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D 0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms. (author)

  20. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Sun Jianxiang; Sun Weijian; Sui Jianli; Zhou Pingkun

    2008-01-01

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60 Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D 0 or SF 2 ). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  1. Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

    Science.gov (United States)

    Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2014-02-01

    Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

  2. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    2010-01-01

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

  3. DNA labeled during phosphonoacetate inhibition and following its reversal in herpesvirus infected cells

    International Nuclear Information System (INIS)

    Jacob, R.J.

    1984-01-01

    Human embryonic lung cells were pre-equilibrated with phosphonoacetate and 32 P orthophosphate label, then infected with phosphonoacetate-sensitive herpes simplex virus (HSV) type 1. Analyses of viral DNA produced in these cells showed the following. i) Viral DNA was synthesized in infected cells exposed to 100 μg of the drug per ml of medium but not in cells exposed to four-fold higher concentrations of the drug. ii) At 300 μg/ml a region of the DNA between 0.58 and 0.69 map units became transiently labeled, but the restriction endonuclease fragment containing these sequences migrated more slowly than the corresponding fragment from virion DNA. iii) Viral DNA extracted from infected cells 1.5 hours post drug withdrawal (300 μg/ml) was preferentially labeled in 2 regions of the genome mapping between 0.17 and 0.23 and 0.58-0.69 map units. This finding is in agreement with a report of Friedman et al. suggesting that HSV DNA contains two different sites if initiation. In addition a 4.8 x 10 6 molecular weight fragment was also preferentially labeled. This fragment could represent a smaller, aberrantly migrating fragment from the 0.17-0.27 map unit region of the DNA. iv) Viral DNA extracted from infected cells at longer intervals after drug withdrawal showed an increasing gradient of radioactivity progressively labeling the genome. These results are consistent with the hypothesis that viral DNA has at least two sites of initiation of DNA synthesis and that both sites are within the L component of the DNA. Alternatively, the results could be interpreted as two sites of localized synthesis (repair) that are detected at high concentrations of phosphonoacetate and immediately following reversal of inhibition of DNA synthesis. The results do not exclude the possibility that secondary sites in both L and S are utilized late in infection or in untreated cells. (Author)

  4. 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.

  5. Cell cycle phase of nondividing cells in aging human cell cultures determined by DNA content and chromosomal constitution

    International Nuclear Information System (INIS)

    Yanishevsky, R.M.

    1975-01-01

    Human diploid cell cultures, strain WI-38, have a finite proliferative capacity and have been proposed as a model of biological aging. To identify the cell cycle phase of the nondividing cells, cultures of various ages were exposed to 3 Hdt for 48 hours to label dividing cells, then the cycle phase was identified for individual cells by one of two methods, and finally, the proliferative status of the same cells was scored by autoradiographic evidence of 3 HdT uptake. The methods to identify the cycle phase were: determination of DNA strain content by Feulgen scanning cytophotometry, and determination of chromosome constitution by the technique of premature chromosome condensation (PCC). Preliminary experiments showed the effect of continuous exposure to various levels of 3 HdT on cell growth. High levels of 3 HdT inhibited cell cycle traverse: the cell number and labeling index curves reached a plateau; the cell volume increased; the cells accumulated with 4C DNA contents and it appeared that they blocked in G 2 phase. This pattern is consistent with a radiation effect. (U.S.)

  6. Committee Opinion No. 640: Cell-Free DNA Screening For Fetal Aneuploidy.

    Science.gov (United States)

    2015-09-01

    Noninvasive prenatal screening that uses cell-free DNA from the plasma of pregnant women offers tremendous potential as a screening method for fetal aneuploidy. A number of laboratories have validated different techniques for the use of cell-free DNA as a screening test for fetal aneuploidy. All tests have a high sensitivity and specificity for trisomy 18 and trisomy 21, regardless of which molecular technique is used. Women whose results are not reported, indeterminate, or uninterpretable (a "no call" test result) from cell-free DNA screening should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy. Patients should be counseled that cell-free DNA screening does not replace the precision obtained with diagnostic tests, such as chorionic villus sampling or amniocentesis and, therefore, is limited in its ability to identify all chromosome abnormalities. Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects. Patients who are undergoing cell-free DNA screening should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment. The cell-free DNA screening test should not be considered in isolation from other clinical findings and test results. Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone. Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy. Given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.

  7. Committee Opinion Summary No. 640: Cell-Free DNA Screening For Fetal Aneuploidy.

    Science.gov (United States)

    2015-09-01

    Noninvasive prenatal screening that uses cell-free DNA from the plasma of pregnant women offers tremendous potential as a screening method for fetal aneuploidy. A number of laboratories have validated different techniques for the use of cell-free DNA as a screening test for fetal aneuploidy. All tests have a high sensitivity and specificity for trisomy 18 and trisomy 21, regardless of which molecular technique is used. Women whose results are not reported, indeterminate, or uninterpretable (a "no call" test result) from cell-free DNA screening should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy. Patients should be counseled that cell-free DNA screening does not replace the precision obtained with diagnostic tests, such as chorionic villus sampling or amniocentesis and, therefore, is limited in its ability to identify all chromosome abnormalities. Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects. Patients who are undergoing cell-free DNA screening should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment. The cell-free DNA screening test should not be considered in isolation from other clinical findings and test results. Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone. Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy. Given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.

  8. 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 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.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.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].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 mechanisms of HIV-1 transmission and help develop more effective

  9. DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    2015-05-01

    Full Text Available Expression of type I interferons (IFNs can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

  10. Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells.

    Directory of Open Access Journals (Sweden)

    P J Hastings

    2010-05-01

    Full Text Available Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s, tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also

  11. Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

    International Nuclear Information System (INIS)

    Bedford, P.; Fichtinger-Schepman, A.M.; Shellard, S.A.; Walker, M.C.; Masters, J.R.; Hill, B.T.

    1988-01-01

    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

  12. Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

    International Nuclear Information System (INIS)

    Brown, T.C.; Boyd, J.B.

    1981-01-01

    Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant. (orig./AJ) [de

  13. PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage

    International Nuclear Information System (INIS)

    Hemstroem, Therese H.; Joseph, Bertrand; Schulte, Gunnar; Lewensohn, Rolf; Zhivotovsky, Boris

    2005-01-01

    Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC

  14. Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells

    Science.gov (United States)

    Tomimatsu, Nozomi; Mukherjee, Bipasha; Burma, Sandeep

    2009-01-01

    The cellular response to DNA double-strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell-cycle-dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell-cycle-independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM-deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM-deficient cells enforce an early G2/M checkpoint that is ATR-dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM-deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP-1), a protein involved in chromatin remodelling, is mediated by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in a spatio-temporal manner in addition to ATM. We posit that ATM substrates involved in cell-cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA-PKcs in addition to ATM. PMID:19444312

  15. Cell cycle-regulated centers of DNA double-strand break repair

    DEFF Research Database (Denmark)

    Lisby, Michael; Antúnez de Mayolo, Adriana; Mortensen, Uffe H

    2003-01-01

    In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms...... of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair....

  16. 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.

  17. Base excision repair of DNA in γ-irradiated human cells

    International Nuclear Information System (INIS)

    Moran, M.F.; Ebisuzaki, Kaney

    1987-01-01

    Escherichia coli endonuclease IV was used to incise cellular DNA specifically at apurinic/apyrimidinic (AP) sites prior to alkaline elution to measure the resulting DNA strand breaks. γ-Irradiated HeLa cells initially contained DNA strand breaks and no AP sites. Upon incubation at 37 0 C the strand breaks were rapidly repaired and AP sites were generated and subsequently repaired. The transient nature of the AP sites indicates the in vivo operation of a base excision repair pathway whereby damaged bases are removed from DNA by DNA glycosylases to produce AP intermediates that are then substrates for AP endonucleases. (author)

  18. 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.

  19. 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 lymphoblast......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...

  20. Postreplication gap filling in the DNA of X-ray-damaged Chinese hamster cells

    International Nuclear Information System (INIS)

    Koerner, I.; Malz, W.

    1975-01-01

    In X-irradiated Chinese hamster cells the newly synthesized DNA has a lower molecular weight than the DNA in control cells. This reduced molecular weight has been interpreted by gap induction opposite the lesions in the parental DNA strands. Within two hours these postreplication gaps were closed. With the aid of BrdUrd-photolys-technique it could be demonstrated that the gaps were filled by de novo synthesis. But we were not able to show a participation of parental DNA in the gap-filling process

  1. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    Science.gov (United States)

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-04-20

    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 recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

  2. Repair capability of mammalian cell fractions demonstrated using infectivity of bacteriophage DNA

    International Nuclear Information System (INIS)

    Lai, S.P.; Lytle, C.D.; Benane, S.G.

    1976-01-01

    Extracts of Potoroo kidney cells (PtK2) were examined for ability to provide a repair function in vitro. The biological activity (infectivity) of uv-irradiated replicative form (RF) DNA of bacteriophage phiX174 was restored during incubation of the DNA with a nuclear extract but not with a cytoplasmic extract. The infectivity of the RF-DNA was determined in spheroplasts of E. coli C/sub s/, which is HCR - . This system for biological assay of uv-irradiated DNA repaired in vitro may be used to complement biochemical and biophysical investigations of molecular repair mechanisms in mammalian cells

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Yue Han

    2013-01-01

    Full Text Available Intrinsic oxidative stress through increased production of reactive oxygen species (ROS is associated with carcinogenic transformation, cell toxicity, and DNA damage. Mitochondrial DNA (mtDNA is a natural surrogate to oxidative DNA damage. MtDNA damage results in the loss of its supercoiled structure and is readily detectable using a novel, supercoiling-sensitive real-time PCR method. Our studies have demonstrated that mtDNA damage, as measured by DNA strand breaks and copy number depletion, is very sensitive to exogenous H2O2 but independent of endogenous ROS production in both prostate cancer and normal cells. In contrast, aggressive prostate cancer cells exhibit a more than 10-fold sensitivity to H2O2-induced cell toxicity than normal cells, and a cascade of secondary ROS production is a critical determinant to the differential response. We propose a new paradigm to account for different mechanisms governing cellular oxidative stress, cell toxicity, and DNA damage with important ramifications in devising new techniques and strategies in prostate cancer prevention and treatment.

  5. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    International Nuclear Information System (INIS)

    Nishiyama, Y.; Rapp, F.

    1981-01-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

  6. Induction of DNA strand breaks in 14C-labelled cells

    International Nuclear Information System (INIS)

    Sundell-Bergman, S.; Johanson, K.J.

    1979-01-01

    Chinese hamster cells grown in vitro were labelled with 14 C-thymidine for 18 hours and after 3 hours in non-radioactive medium they were stored at 0 0 C for various periods ( 1 to 12 hours). During this treatment a number of DNA strand breaks were induced by 14 C decay which were not repaired at 0 0 C. The number of DNA strand breaks was determined using the DNA unwinding technique. At 0.5-1 dpm per cell a detectable number of DNA strand breaks were found. Treatment for six hours (1 dpm per cell) reduced the percentage of double-stranded DNA from 80 to 70%, corresponding to about 750 DNA strand breaks per cell. The rejoining of DNA strand breaks was studied after treatment for 12 hours at 0 0 C followed by incubation of the cells for various periods at 37 0 C. Most of the DNA strand breaks induced by 14 C decay at 0 0 C were repaired after incubation at 37 0 C for 15 minutes. Assuming an absorbed dose of 1.8 mGy per 14 C decay to the cell nucleus an RBE value close to 1 was found for internal irradiation from 14 C decay as compared with 60 Co-gamma irradiation. (author)

  7. Cell cycle-regulated centers of DNA double-strand break repair

    DEFF Research Database (Denmark)

    Lisby, Michael; Antúnez de Mayolo, Adriana; Mortensen, Uffe H

    2003-01-01

    In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms...... foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends...... of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair....

  8. Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

    Science.gov (United States)

    Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

    2014-01-01

    The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

  9. Controlling kinase activity during the cell cycle: from the DNA damage response to mitosis

    NARCIS (Netherlands)

    Bruinsma, W.

    2014-01-01

    The cell cycle is the process through which cells execute cell division. This is essential for many basal processes such as organismal development, tissue maintenance and reproduction. Disruption of the cell cycle, for example by damaged DNA, can have vast consequences and can lead to many diseases

  10. B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization.

    Science.gov (United States)

    Fontoura, I C; Trombone, A P F; Almeida, L P; Lorenzi, J C C; Rossetti, R A M; Malardo, T; Padilha, E; Schluchting, W; Silva, R L L; Gembre, A F; Fiuza, J E C; Silva, C L; Panunto-Castelo, A; Coelho-Castelo, A A M

    2015-12-01

    In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

  11. The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

    International Nuclear Information System (INIS)

    Klein, W.; Altmann, H.; Kocsis, F.; Egg, D.; Guenther, R.

    1978-03-01

    ''In vitro'' experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A 1 , B 1 , E 1 , E 2 and Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE 1 and PgE 2 at 5 ng/ml and at 2,5 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE 1 , PgE 2 and PgF 2 at 2,5 μg/ml. (author)

  12. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    Directory of Open Access Journals (Sweden)

    M Emmy M Dolman

    Full Text Available Tumor cells might resist therapy with ionizing radiation (IR by non-homologous end-joining (NHEJ of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK. The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  13. Effects of ionizing radiations on DNA replication in cultured mammalian cells

    International Nuclear Information System (INIS)

    Makino, F.; Okada, S.

    1975-01-01

    The dose-response curve of [ 3 H] thymidine incorporation into the acid-insoluble fraction of cultured mammalian cells, grown in the presence of 10 -4 M cold thymidine, is different from that of incorporation in the absence of cold thymidine. For quantitative estimation of net DNA synthesis in nonirradiated and irradiated cells, two methods were used: isolation of newly synthesized BUdR-labeled DNA by CsCl gradient centrifugation and a fluorometric estimation of DNA content in the synchronized population. Both methods showed that the depression of [ 3 H]thymidine incorporation in the presence of cold thymidine reflected a depression of net DNA synthesis. Radiosensitive steps in DNA synthesis were examined by the use of alkaline sucrose gradient centrifugation. The rate of replication along the DNA strands was inhibited to a lesser extent than that of over-all DNA synthesis. The labeling patterns of DNA exposed to [ 3 H]thymidine for 20 min indicated that ionizing radiation preferentially interfered with the formation of small-size 3 H-labeled DNA pieces. These results suggest that the initiation of DNA replication is more radiosensitive than the elongation of DNA strands whose replication has already been initiated. (U.S.)

  14. Cell-free DNA as a post-treatment surveillance strategy: current status.

    Science.gov (United States)

    Burgener, Justin M; Rostami, Ariana; De Carvalho, Daniel D; Bratman, Scott V

    2017-10-01

    Circulating tumor DNA (ctDNA) consists of cell-free DNA (cfDNA) fragments that are released from tumor cells into the bloodstream. ctDNA harbors cancer-specific genetic and epigenetic alterations that allow its detection and quantification using a variety of emerging techniques. The promise of convenient non-invasive access to the complex and dynamic molecular features of cancer through peripheral blood has galvanized translational researchers around this topic with compelling routes to clinical implementation, particularly in the post-treatment surveillance setting. Although analysis methods must contend with the small quantities of ctDNA present in most patients, and the relative over-abundance of background cfDNA derived from normal tissues, recent technical innovations have led to dramatic improvements in the sensitivity of ctDNA detection. As a result, ever more studies are investigating the clinical utility of ctDNA for applications in (1) treatment response assessment, (2) identification of emerging resistance mechanisms, (3) minimal residual disease detection, and (4) characterization of clonal heterogeneity and selection. In this review, we describe the detection methods currently used in clinical studies to assess low fractions of ctDNA, as well as their utility in the applications previously described. Finally, we address current limitations that have hampered the clinical implementation of ctDNA analysis for post-treatment surveillance and propose steps that could be made to address them. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy

    Science.gov (United States)

    Mieruszynski, Stephen; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

    2015-01-01

    The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell. PMID:26354725

  16. 1Restoration of ATM expression in DNA-PKcs deficient cells inhibits signal end joining

    Science.gov (United States)

    Neal, Jessica A.; Xu, Yao; Abe, Masumi; Hendrickson, Eric; Meek, Katheryn

    2016-01-01

    Unlike most DNA-PKcs deficient mouse cell strains, we show here that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those studied here) and in DNA-PKcs deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the “classical” non-homologous end-joining pathway. PMID:26921311

  17. 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.

  18. Protection of tobacco cells from oxidative copper toxicity by catalytically active metal-binding DNA oligomers.

    Science.gov (United States)

    Iwase, Junichiro; Furukawa, Hiroka; Hiramatsu, Takuya; Bouteau, François; Mancuso, Stefano; Tanaka, Kenichiro; Okazaki, Toshihiko; Kawano, Tomonori

    2014-03-01

    The impact of copper ions on the oxidative and calcium signal transductions, leading to cell death in plant cells, have been documented. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species and the stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations. These early events, completed within a few minutes after the contact with copper, are known to trigger the development of cell death. The effects of DNA fragments with copper-binding motifs as novel plant cell-protecting agents were assessed using cell suspension cultures of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. The addition of GC-rich double-stranded DNA fragments, prior to the addition of copper ions, effectively blocked both the copper-induced calcium influx and cell death. In addition, the DNA-Cu complex examined was shown to possess superoxide-scavenging catalytic activity, suggesting that DNA-mediated protection of the cells from copper toxicity is due to the removal of superoxide. Lastly, a possible mechanism of DNA-Cu interaction and future applications of these DNA fragments in the protection of plant roots from metal toxicity or in aid of phyto-remediation processes are discussed.

  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. TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

    KAUST Repository

    Meller, Stephan

    2015-07-13

    Interleukin 17-producing helper T cells (TH 17 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. © 2015 Nature America, Inc.

  1. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    Yatani, Ryuichi; Tohgo, Yukihiro; Kunishima, Nobuyoshi.

    1975-01-01

    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.)

  2. Modern problems of DNA repair in mammalian cells and some unsettled questions

    International Nuclear Information System (INIS)

    Gaziev, A.I.

    1978-01-01

    A comparison of DNA repair process in the cells of mammals and E. coli revealed no principal differences in the enzymic mechanisms of DNA repair in the cells of higher and lower organisms. It has been found that when given is the same number of impairments in the section of DNA chain in the cells of mammals and bacteria the regeneration in the former occurs more slowly than in the latter. Low rate elimination of impairments of DNA in the cells of mammals is due to a more complex intracellular and permolecular organization. It is stressed that the investigation into the mechanisms of fixing impairments in case of postreplication DNA repair is a very important and unresolved problem, especially in terms of radiation mutagenesis and cancerogenesis. Much thought is given to the problem of repairing double stranded ruptures of DNA. It is proposed that DNA repair should be considered not only in terms of functioning of enzymes in DNA metabolism, but also permolecular organization of genome in the cell

  3. 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.

  4. Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Luca A Petruccelli

    Full Text Available Histone deacetylase inhibitors (HDACi are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

  5. Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

    Science.gov (United States)

    Pettersson, Filippa; Retrouvey, Hélène; Skoulikas, Sophia; Miller, Wilson H.

    2011-01-01

    Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. PMID:21695163

  6. 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 effect on the proportion of cell-free DNA (cfDNA) arising from the fetus (fetal fraction). METHODS: Nine pregnant women carrying male fetuses at gestational age 12(+0)  weeks to 14(+6)  weeks were included. Plasma from nine pregnant women was drawn prior to, immediately after, and 30 min after 30 min...... 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 

  7. Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Hansen, Jakob Werner; Kristensen, Søren Sommer

    2016-01-01

    III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after......BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients...... treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter. CONCLUSIONS: Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating...

  8. Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

    International Nuclear Information System (INIS)

    Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

    2003-01-01

    Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

  9. A DNA delivery system targeting dendritic cells for use in ...

    African Journals Online (AJOL)

    DNA-based vaccination has emerged as a promising method of immunisation since the first demonstration of this technology. Improving the antibody responses is desirable for the protective efficacy and hence broad application of these vaccines. We examined the immunogenicity of a Plasmodium-based DNA vaccine that ...

  10. Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons.

    Science.gov (United States)

    Tan, Carlyn Rose C; Zhou, Lanlan; El-Deiry, Wafik S

    2016-06-01

    Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are emerging noninvasive multifunctional biomarkers in liquid biopsy allowing for early diagnosis, accurate prognosis, therapeutic target selection, spatiotemporal monitoring of metastasis, as well as monitoring response and resistance to treatment. CTCs and ctDNA are released from different tumor types at different stages and contribute complementary information for clinical decision. Although big strides have been taken in technology development for detection, isolation and characterization of CTCs and sensitive and specific detection of ctDNA, CTC-, and ctDNA-based liquid biopsies may not be widely adopted for routine cancer patient care until the suitability, accuracy, and reliability of these tests are validated and more standardized protocols are corroborated in large, independent, prospectively designed trials. This review covers CTC- and ctDNA-related technologies and their application in colorectal cancer. The promise of CTC-and ctDNA-based liquid biopsies is envisioned.

  11. Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.

    Science.gov (United States)

    Letendre, Jo-Annie; Goggs, Robert

    2017-05-01

    To determine if cell-free DNA (cfDNA) was identifiable in canine plasma, to evaluate 3 techniques for the measurement of plasma cfDNA concentrations in dogs presented to an emergency service, and to compare the plasma cfDNA concentrations of healthy dogs to those with sepsis, trauma, and neoplasia. Retrospective study of banked canine plasma samples collected between May 2014 and December 2014. Dogs presented to the emergency service of a university veterinary teaching hospital. Plasma cfDNA was measured on residual plasma samples obtained from 15 dogs with sepsis, 15 dogs with moderate-severe trauma, 15 dogs diagnosed with a sarcoma. Plasma cfDNA was also measured in 15 healthy dogs. None. Assay linearity, repeatability, and reproducibility were evaluated. Quantification of cfDNA was performed in duplicate on diluted citrated plasma and following DNA purification using 2 fluorescence assays (SYBR-Gold; Quant-iT) and by ultraviolet absorbance spectroscopy. Fluorescence intensities (FIs) were converted to cfDNA concentrations using standard curves. Median FI values and cfDNA concentrations were compared to healthy controls using the Kruskal-Wallis test, with adjustment for multiple comparisons. Alpha was set at 0.05. Both assays had excellent linearity, and acceptable repeatability and reproducibility. Compared to controls, plasma cfDNA concentrations were significantly increased in dogs with sepsis or moderate-severe trauma with both assays (P ≤ 0.003). Dogs with neoplasia had significantly increased cfDNA concentrations with the Quant-iT assay only (P = 0.003). When measurements were performed on purified DNA, only dogs with moderate-severe trauma had significantly increased cfDNA concentrations (P plasma using 2 fluorescence assays. DNA extraction offers no advantage over direct measurement. Compared to healthy controls, dogs with sepsis or moderate-severe trauma have significantly increased plasma cfDNA concentrations. © Veterinary Emergency and Critical Care

  12. Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA.

    Science.gov (United States)

    Worm Ørntoft, Mai-Britt; Jensen, Sarah Østrup; Hansen, Thomas Birkballe; Bramsen, Jesper Bertram; Andersen, Claus Lindbjerg

    2017-08-01

    Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. To compare cfDNA recovery of different BSC methods, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only 3 kits recovered DNA fragments of BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

  13. Degradation of DNA in Haemophilus influenzae cells after x-ray irradiation. I. Experimental results

    International Nuclear Information System (INIS)

    Randolph, M.L.; Setlow, J.K.

    1975-01-01

    Sequential measurements of DNA in Haemophilus influenzae cells after x-ray irradiation show rapid initial degradation of DNA followed by a plateau after about 40 min at normal growth conditions. Both the initial rate and final amount of degradation increase with radiation exposure. Degradation is somewhat greater in stationary-phase than in log-phase cells, but colony-forming ability (CFA) is independent of cell stage. Distributions of single-strand lengths of DNA in unirradiated or irradiated cases, as measured by alkaline sucrose gradient techniques, are neither monodispersive nor random, and possible causes for nonrandomness are discussed

  14. Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process.

    Science.gov (United States)

    Rodríguez, Alfredo; Torres, Leda; Juárez, Ulises; Sosa, David; Azpeitia, Eugenio; García-de Teresa, Benilde; Cortés, Edith; Ortíz, Rocío; Salazar, Ana M; Ostrosky-Wegman, Patricia; Mendoza, Luis; Frías, Sara

    2015-09-18

    The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage. We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot. Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA. Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express

  15. Cell cycle stage dependent variations in drug-induced topoisomerase II mediated DNA cleavage and cytotoxicity

    International Nuclear Information System (INIS)

    Estey, E.; Adlakha, R.C.; Hittelman, W.N.; Zwelling, L.A.

    1987-01-01

    The DNA cleavage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in mammalian cells is putatively mediated by topoisomerase II. The authors found that in synchronized HeLa cells the frequency of such cleavage was 4-15-fold greater in mitosis than in S while the DNA of G 1 and G 2 cells exhibited an intermediate susceptibility to cleavage. The hypersensitivity of mitotic DNA to m-AMSA-induced cleavage was acquired relatively abruptly in late G 2 and was lost similarly abruptly in early G 1 . The susceptibility of mitotic cells to m-AMSA-induced DNA cleavage was not clearly paralleled by an increase in topoisomerase II activity in 350 mM NaCl extracts from mitotic cells compared to similar extracts from cells in G 1 , S, or G 2 . Furthermore, equal amounts of decatenating activity from cells in mitosis and S produced equal amounts of m-AMSA-induced cleavage of simian virus 40 (SV40) DNA; i.e., the interaction between m-AMSA and extractable enzyme was similar in mitosis and S. The DNA of mitotic cells was also hypersensitive to cleavage by 4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-β-D-glucopyranoside) (etoposide), a drug that produces topoisomerase II mediated DNA cleavage without binding to DNA. Cell cycle stage dependent fluctuations in m-AMSA-induced DNA cleavage may result from fluctuations in the structure of chromatin per se that occur during the cell cycle. Surprisingly, cell cycle stage dependent differences in m-AMSA-induced DNA cleavage did not correlate with differences in the susceptibility to the cytotoxic effects of the drug. In fact, cells in S were most sensitive to these effects. These results are an exception to the previously observed parallel between the susceptibility of mammalian cells to drug-induced DNA cleavage and the susceptibility of the cells to drug-induced cytotoxicity and indicate the complexity of any relationship between the two phenomena

  16. The first successful observation of in-cell NMR signals of DNA and RNA in living human cells.

    Science.gov (United States)

    Yamaoki, Yudai; Kiyoishi, Ayaka; Miyake, Masayuki; Kano, Fumi; Murata, Masayuki; Nagata, Takashi; Katahira, Masato

    2018-01-31

    In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.

  17. Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR.

    Science.gov (United States)

    Ye, Wei; Tang, Xiaojun; Liu, Chu; Wen, Chaowei; Li, Wei; Lyu, Jianxin

    2017-04-01

    To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ 0 ) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA. In the results, the ddPCR method showed high sensitivity and specificity. When the DNA was extracted from plasma prior to ddPCR, the ccf-mtDNA copy number was higher than that measured without extraction. This difference was not due to a PCR inhibitor, such as EDTA-Na 2 , an anti-coagulant in plasma, because standard EDTA-Na 2 concentration (5 mM) did not significantly inhibit ddPCR reactions. The difference might be attributable to plasma exosomal mtDNA, which was 4.21 ± 0.38 copies/μL of plasma, accounting for ∼19% of plasma ccf-mtDNA. Therefore, ddPCR can quickly and reliably detect ccf-mtDNA from plasma with a prior DNA extraction step, providing for a more accurate detection of ccf-mtDNA. The direct use of plasma as a template in ddPCR is suitable for the detection of exogenous cell-free nucleic acids within plasma, but not of nucleic acids that have a vesicle-associated form, such as exosomal mtDNA. Graphical Abstract Designs of the present work. *: Module 1, #: Module 2, &: Module 3.

  18. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.

    Directory of Open Access Journals (Sweden)

    Sabine S Lange

    2016-01-01

    Full Text Available DNA polymerase ζ (pol ζ is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.

  19. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.

    Science.gov (United States)

    Lange, Sabine S; Tomida, Junya; Boulware, Karen S; Bhetawal, Sarita; Wood, Richard D

    2016-01-01

    DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.

  20. Relationships between Cell-Free DNA and Serum Analytes in First and Second Trimesters of Pregnancy

    Science.gov (United States)

    Vora, Neeta L.; Johnson, Kirby L.; Lambert-Messerlian, Geralyn; Tighiouart, Hocine; Peter, Inga; Urato, Adam C.; Bianchi, Diana W.

    2010-01-01

    Objective Circulating cell-free DNA and maternal serum analytes are indicators of fetal and placental condition. Little is known about the relationship of these noninvasive markers to each other, particularly in the first trimester. Our goal was to assess the relationship between first and second trimester cell-free DNA levels and maternal serum screening markers. Methods First and second trimester residual maternal serum samples from 50 women were obtained. First trimester (pregnancy-associated plasma protein A [PAPP-A] and β-hCG), and second trimester serum analytes (β-hCG, alpha-fetoprotein [AFP], unconjugated estriol and inhibin A) had been measured at the time of sample receipt. All fetuses were male, as confirmed by birth records. Cell-free DNA was extracted and measured by real-time quantitative polymerase chain reaction (PCR) amplification using glyceraldehyde phosphate dehydrogenase (GAPDH) and DYS1 as markers of total DNA and fetal DNA, respectively. Determination of linear associations between first and second trimester serum markers and cell-free DNA levels using Pearson correlations was performed. Results Statistically significant correlations between first trimester PAPP-A multiples of the median (MoMs) and both total (r=0.36, p=0.016) and fetal (r= 0.41, p=0.006) DNA in the first trimester were observed. There were no significant correlations between first trimester serum hCG or any second trimester serum marker with DNA levels. Conclusions Correlation between serum PAPP-A and first trimester circulating cell-free fetal and total DNA levels is a novel finding. PAPP-A is a glycoprotein of placental origin, and its correlation to cell-free fetal DNA in maternal serum suggests a common tissue origin, through apoptosis of placental cells. However, since PAPP-A and cell-free DNA were only marginally correlated and cell-free DNA can be reliably detected in the first trimester, the addition of cell-free DNA to serum screening strategies may be helpful in

  1. Purification of DNA for the transfection of a Spodoptera frugiperda cell line.

    Science.gov (United States)

    Slack, Jeffrey M; Lawrence, Susan D

    2002-01-01

    Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.

  2. DNA methylation alterations induced by transient exposure of MCF-7 cells to maghemite nanoparticles.

    Science.gov (United States)

    Bonadio, Raphael S; Arcanjo, Ana Carolina; Lima, Emilia Cd; Vasconcelos, Alline T; Silva, Renata C; Horst, Frederico H; Azevedo, Ricardo B; Poças-Fonseca, Marcio José; F Longo, João Paulo

    2017-12-01

    To evaluate the DNA methylation profile of MCF-7 cells during and after the treatment with maghemite nanoparticles (MNP-CIT). Noncytotoxic MNP-CIT concentrations and cell morphology were evaluated by standard methods. DNA methylation was assessed by whole genome bisulfite sequencing. DNA methyltransferase (DNMT) genes expression was analyzed by qRT-PCR. A total of 30 and 60 µgFeml -1 MNP-CIT accumulated in cytoplasm but did not present cytotoxic effects. The overall percentage of DNA methylation was not affected, but 58 gene-associated regions underwent DNA methylation reprogramming, including genes related to cancer onset. DNMT transcript levels were also modulated. Transient exposure to MNP-CIT promoted epigenomic changes and altered the DNMT genes regulation in MCF-7 cells. These events should be considered for biomedical applications.

  3. Recovery of CHO cells from hyperthermic potentiation to x rays: repair of DNA and chromatin

    International Nuclear Information System (INIS)

    Clark, E.P.; Dewey, W.C.; Lett, J.T.

    1981-01-01

    Above the critical temperature, ca. 42.5 0 C, hyperthermic potentiation of Chinese hamster ovary (CHO) cells to x irradiation was accompanied by increased binding of nonhistone proteins to DNA and by reduced rates of rejoining of DNA strand breaks. These biochemical changes were reversed as the cells recovered from the hyperthermic exposures at 37 0 C. If the hyperthermically treated cells were incubated at 37 0 C before x irradiation, the ratio of nonhistone protein to DNA returned to normal in 12 h but the depressed rate of rejoining of DNA strand breaks and increased cell radiosensitivity remained unaltered. Cell radiosensitivity began to decrease after 12 h and recovery from hyperthermia-potentiated radiosensitivity was complete by 48 h. In the same interval, the rate of rejoining of DNA strand breaks also returned to normal. From this behavior, we conclude that the reduction in the rate of rejoining of DNA strand breaks involved changes in DNA structure which were restored only after the thermal enhancement of protein binding was reversed. These experiments provide support for the viewpoint that critical hyperthermic potentiation (i.e., above 42.5 0 C for CHO cells) may have logistical advantages over subcritical hyperthermic potentiation (i.e., below 42.5 0 C) in clinical situations

  4. Heterogeneity in white blood cells has potential to confound DNA methylation measurements.

    Directory of Open Access Journals (Sweden)

    Bjorn T Adalsteinsson

    Full Text Available Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179 and in two white blood cell fractions (n = 20, isolated using density gradient centrifugation, in four CGIs (CpG Islands located in genes HHEX (10 CpG sites assayed, KCNJ11 (8 CpGs, KCNQ1 (4 CpGs and PM20D1 (7 CpGs. Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter explained up to 40% (p<0.0001 of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

  5. Cell cycle regulation of DNA polymerase beta in rotenone-based Parkinson's disease models.

    Directory of Open Access Journals (Sweden)

    Hongcai Wang

    Full Text Available In Parkinson's disease (PD, neuronal cells undergo mitotic catastrophe and endoreduplication prior to cell death; however, the regulatory mechanisms remain to be defined. In this study, we investigated cell cycle regulation of DNA polymerase β (poly β in rotenone-based dopaminergic cellular and animal models. Incubation with a low concentration (0.25 µM of rotenone for 1.5 to 7 days resulted in a flattened cell body and decreased DNA replication during S phase, whereas a high concentration (2 µM of rotenone exposure resulted in enlarged, multi-nucleated cells and converted the mitotic cycle into endoreduplication. Consistently, DNA poly β, which is mainly involved in DNA repair synthesis, was upregulated to a high level following exposure to 2 µM rotenone. The abrogation of DNA poly β by siRNA transfection or dideoxycytidine (DDC treatment attenuated the rotenone-induced endoreduplication. The cell cycle was reactivated in cyclin D-expressing dopaminergic neurons from the substantia nigra (SN of rats following stereotactic (ST infusion of rotenone. Increased DNA poly β expression was observed in the substantia nigra pars compacta (SNc and the substantia nigra pars reticulate (SNr of rotenone-treated rats. Collectively, in the in vitro model of rotenone-induced mitotic catastrophe, the overexpression of DNA poly β promotes endoreduplication; in the in vivo model, the upregulation of DNA poly β and cell cycle reentry were also observed in the adult rat substantia nigra. Therefore, the cell cycle regulation of DNA poly β may be involved in the pathological processes of PD, which results in the induction of endoreduplication.

  6. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Helmig, Susanne; Zahn, Daniela; Kubiak, Thomas; Michal, Matthias; Gori, Tommaso; Ehlert, Tobias; Beiter, Thomas; Simon, Perikles

    2014-01-01

    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.

  7. 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.

  8. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    International Nuclear Information System (INIS)

    Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

    1984-01-01

    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

  9. Circulating cell-free DNA levels increase variably following chorionic villus sampling.

    Science.gov (United States)

    Vora, Neeta L; Johnson, Kirby L; Peter, Inga; Tighiouart, Hocine; Ralston, Steven J; Craigo, Sabrina D; Bianchi, Diana W

    2010-04-01

    Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA. Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender. Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels. While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women. Copyright (c) 2010 John Wiley & Sons, Ltd.

  10. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    International Nuclear Information System (INIS)

    Maltas, Esra; Malkondu, Sait; Uyar, Pembegul; Ozmen, Mustafa

    2015-01-01

    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

  11. 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

  12. DNA repair in human cells: Methods for the determination of calmodulin involvement

    International Nuclear Information System (INIS)

    Charp, P.A.

    1987-01-01

    Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

  13. Construction of porcine CCK pDNA and its expression in COS-7 cells.

    Science.gov (United States)

    Bai, Jigang; Lü, Yi; Bai, Qiaoling

    2007-06-01

    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 lepofectamin 2000-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.

  14. Nano-ranged low-energy ion-beam-induced DNA transfer in biological cells

    International Nuclear Information System (INIS)

    Yu, L.D.; Wongkham, W.; Prakrajang, K.; Sangwijit, K.; Inthanon, K.; Thongkumkoon, P.; Wanichapichart, P.; Anuntalabhochai, S.

    2013-01-01

    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.

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

    International Nuclear Information System (INIS)

    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 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 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

  16. 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.

  17. DNA Damage in CD133-Positive Cells in Barrett’s Esophagus and Esophageal Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Raynoo Thanan

    2016-01-01

    Full Text Available Barrett’s esophagus (BE caused by gastroesophageal reflux is a major risk factor of Barrett’s esophageal adenocarcinoma (BEA, an inflammation-related cancer. Chronic inflammation and following tissue damage may activate progenitor cells under reactive oxygen/nitrogen species-rich environment. We previously reported the formation of oxidative/nitrative stress-mediated mutagenic DNA lesions, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG and 8-nitroguanine, in columnar epithelial cells of BE tissues and cancer cells of BEA tissues. We investigated the mechanisms of BEA development in relation to oxidative/nitrative DNA damage and stem cell hypothesis. We examined 8-nitroguanine and 8-oxodG formation and the expression of stem cell marker (CD133 in biopsy specimens of patients with BE and BEA by immunohistochemical analysis in comparison with those of normal subjects. CD133 was detected at apical surface of columnar epithelial cells of BE and BEA tissues, and the cytoplasm and cell membrane of cancer cells in BEA tissues. DNA lesions and CD133 were colocalized in columnar epithelial cells and cancer cells. Their relative staining intensities in these tissues were significantly higher than those in normal subjects. Our results suggest that BE columnar epithelial cells with CD133 expression in apical surface undergo inflammation-mediated DNA damage, and mutated cells acquire the property of cancer stem cells with cytoplasmic CD133 expression.

  18. 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

  19. 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…

  20. Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    Pamela Mehanna

    Full Text Available Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

  1. p53 Specifically Binds Triplex DNA In Vitro and in Cells

    NARCIS (Netherlands)

    Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

    2016-01-01

    Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA

  2. P53 specifically binds triplex DNA in vitro and in cells

    NARCIS (Netherlands)

    Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

    2016-01-01

    Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA

  3. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    Lohrer, H.

    1987-01-01

    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.) [de

  4. Differential role of eDNA, proteins, and polysaccharides in cell-cell and cell-substrate adhesion by three Staphylococcus species

    DEFF Research Database (Denmark)

    Meyer, Rikke Louise; Okshevsky, Mira Ursula; Zeng, Guanghong

    valuable for designing new approaches to biofilm prevention. In this study, we combine microfluidic flow-cell studies with single-cell analyses to understand how polysaccharides, extracellular DNA (eDNA), and proteins contribute individually and in concert to mediate bacterial adhesion and aggregation...... on abiotic surfaces. We quantified initial adhesion, cell aggregation, and single-cell adhesion forces of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus xylosus in the presence and absence of DNase, dispersin, or subtilisin, which cleave extracellular DNA, polysaccharides and proteins...... affected by DNase and dispersin treatments, hence eDNA and polysaccharides were essential for cell-cell interactions. We showed that proteins, polysaccharides and eDNA contribute differently to the adhesion of three Staphylcococcus species, underlining the need to either tailor biofilm prevention...

  5. 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.

  6. Radiation-produced electron migration along 5-bromouracil-substituted DNA in cells and in solutions

    International Nuclear Information System (INIS)

    Beach, C.M.

    1981-01-01

    Results of work by other investigators support the theory of charge migration in DNA. Charge transfer between nucleotides and electron and energy migration in solid state DNA have been detected, but no previous experiments have demonstrated charge migration in aqueous solutions of DNA or in DNA inside an E. coli cell. Such experiments were performed by substituting different amounts of 5-bromouracil (BU) for thymine in E. coli DNA and assaying for the amount of bromide given off from the reaction of bromouracil with hydrated electrons produced by ionizing radiation to form uracil-5-yl radicals and free bromide. By varying the amount of BU incorporated in the DNA, the average distance between the BU bases was varied, and because the number of BU/electron reactions was monitored by the amount of bromide released, the maximum average electron migration distance along the BU-DNA was estimated. Hydrated electrons, e/sub aq/, were shown to react with BU in BU-DNA with the resultant release of bromide with G(-BR - ) = 0.519 +- 0.062. OH radicals were half as reactive as e/sub aq/ toward producing bromide from BU-DNA. O 2 , which has been shown to transfer charge to BU in aqueous solution, did not transfer charge to BU-DNA. The CO 2 radical was shown to cause the release of bromide from BU-DNA at least as effectively as e/sub aq/. Charge migration was demonstrated, and the maximum average electron migration distance in aqueous solutions of BU-DNA was measured to be 8 to 10 base distances (assuming only intrastrand migration). Only 11% to 16% of the electrons produced attacked BU-DNA in aqueous solution, and only 1% resulted in bromide release from BU-DNA inside E. coli. Charge migration was demonstrated in BU-DNA inside E. coli., and the maximum average migration distance was measured to be 5 to 6 base distances

  7. UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein

    International Nuclear Information System (INIS)

    Carty, M.P.; Zernik-Kobak, M.; McGrath, S.; Dixon, K.

    1994-01-01

    We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus. (Author)

  8. 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.

  9. Presence of UV-endonuclease sensitive sites in daughter DNA of UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    D'Ambrosio, S.; Setlow, R.B.

    1978-02-01

    Asynchronous Chinese hamster cells were irradiated with 10 Jm -2 uv radiation and 0.25 to 4 hours later pulse-labeled with [ 3 H]thymidine. Cells synchronized by shaking off mitotic and G 1 cells were irradiated in either the G 1 -phase or S-phase of the cell cycle and pulse-labeled with [ 3 H]thymidine in the S-phase. After a 12 to 14 hour chase in unlabeled medium, the DNA was extracted, incubated with Micrococcus luteus uv-endonuclease and sedimented in alkaline sucrose. The number of endonuclease sensitive sites decreased as the time between uv irradiation and pulse-labeling of daughter DNA increased. Further, there were significantly less endonuclease sensitive sites in the daughter DNA from cells irradiated in the G 1 -phase than in the S-phase. These data indicate that very few, if any, dimers are transferred from parental DNA to daughter DNA and that the dimers detected in daughter DNA may be due to the irradiation of replicating daughter DNA before labeling

  10. Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.

    1986-01-01

    The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

  11. Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Clark, J.M.; Hanawalt, P.C.

    1982-07-01

    The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with /sup 3/H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Lori E. Lowes

    2016-09-01

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction...

  16. DNA repair in Bacillus subtilis: excision repair capacity of competent cells

    International Nuclear Information System (INIS)

    Yasbin, R.E.; Fernwalt, J.D.; Fields, P.I.

    1979-01-01

    Competent Bacillus subtilis were investigated for their ability to support the repair of uv-irradiated bacteriophage and bacteriophage DNA. uv-irradiated bacteriophage DNA cannot be repaired to the same level as uv-irradiated bacteriophage, suggesting a deficiency in the ability of competent cells to repair uv damage. However, competent cells were as repair proficient as noncompetent cells in their ability to repair irradiated bacteriophage in marker rescue experiments. The increased sensitivity of irradiated DNA is shown to be due to the inability of excision repair to function on transfecting DNA in competent bacteria. Furthermore, competent cells show no evidence of possessing an inducible BsuR restriction system to complement their inducible BsuR modification enzyme

  17. A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    2017-11-01

    Full Text Available A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.

  18. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    Science.gov (United States)

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  19. ATP-independent DNA synthesis in Vaccinia-infected L cells

    International Nuclear Information System (INIS)

    Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

    1978-01-01

    Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

  20. UV-induced unscheduled DNA synthesis in cultured human epidermal and dermal cells

    International Nuclear Information System (INIS)

    Hosomi, Jiro; Kuroki, Toshio

    1985-01-01

    DNA repair in human epidermal cells, a target of skin carcinogenesis, was examined by measuring unscheduled DNA synthesis on autoradiographs. Epidermal cells were obtained from normal subjects and all experiments were performed on primary cultures. UV-irradiation induced unscheduled DNA synthesis in human epidermal cells dose-dependently at doses of 5 to 20 J/m 2 . The range of induction varied 3-fold in 9 cultures derived from different donors. No correlation was found between the extent of unscheduled DNA synthesis and the age or sex of the donors. For comparison, human dermal fibroblasts and mouse epidermal and dermal cells were examined under the same conditions. In human dermal cells, the number of grains was about 3.3 times that in epidermal cells. Mouse epidermal cells isolated from newborn C3H/He and Sencar mice showed almost the same extent of unscheduled DNA synthesis as human cells, but the response of dermal fibroblasts of mice was about 2 to 3 times less than that of human fibroblasts. The relevance of these differences among individuals, cell types and species is discussed. (author)

  1. Developing DNA vaccines that call to dendritic cells

    OpenAIRE

    Kutzler, Michele A.; Weiner, David B.

    2004-01-01

    DNA vaccination is a novel immunization strategy that has great potential for the development of vaccines and immune therapeutics. This strategy has been highly effective in mice, while less immunogenic in nonhuman primates and humans. Enhancing DNA vaccine potency remains a challenge. It is likely that APCs, and especially DCs, play a paramount role in the presentation of vaccine antigen to the immune system. A new study reports the synergistic recruitment, expansion, and activation of DCs i...

  2. Amifostine does not protect thyroid cancer cells in DNA damaging in vitro models

    Directory of Open Access Journals (Sweden)

    Joanna Klubo-Gwiezdzinska

    2017-09-01

    Full Text Available Background: Amifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells. Methods: We investigated the effects of the active form of amifostine (WR-1065 on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643 and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM, by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL and by γ-radiation (6 Gy. DNA damage, cell viability and apoptosis were examined. Results: We demonstrated the selective action of WR-1065 (0.1 mM, which prevented oxidative stress–induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis. Conclusions: Our data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.

  3. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer.

    Science.gov (United States)

    Burgstaller, Jörg P; Schinogl, Pamela; Dinnyes, Andras; Müller, Mathias; Steinborn, Ralf

    2007-12-21

    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. 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. 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 tested, whereby all but one case revealed less

  4. 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

  5. Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus derived cDNA expression vector.

    NARCIS (Netherlands)

    P.B.G.M. Belt; W. Jongmans; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude); P. van de Putte (Pieter)

    1991-01-01

    textabstractHuman cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this

  6. DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

    Science.gov (United States)

    Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

    2017-11-15

    DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    OpenAIRE

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    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...

  8. Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.

    1971-01-01

    Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal

  9. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

    Science.gov (United States)

    Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-12-22

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

  10. Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

    Science.gov (United States)

    Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

    2017-11-01

    Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Identification of potentially cytotoxic lesions induced by UVA photoactivation of DNA 4-thiothymidine in human cells.

    Science.gov (United States)

    Reelfs, Olivier; Macpherson, Peter; Ren, Xiaolin; Xu, Yao-Zhong; Karran, Peter; Young, Antony R

    2011-12-01

    Photochemotherapy-in which a photosensitizing drug is combined with ultraviolet or visible radiation-has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S(4)TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S(4)TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S(4)TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S(4)TdR in dilute solution, more complex lesions are formed when S(4)TdR-containing oligonucleotides are irradiated. One of these, a thietane/S(5)-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S(4)TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S(4)TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S(4)TdR/UVA indicating that these lesions contribute significantly to S(4)TdR/UVA cytotoxicity. © The Author(s) 2011. Published by Oxford University Press.

  12. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    Science.gov (United States)

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  13. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA from Patients with Non-Small Cell Lung Cancer (NSCLC.

    Directory of Open Access Journals (Sweden)

    James L Sherwood

    Full Text Available Non-invasive mutation testing using circulating tumour DNA (ctDNA is an attractive premise. This could enable patients without available tumour sample to access more treatment options.Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.2 hr incubation time and double plasma centrifugation (2000 x g reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA. Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT (Streck, after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

  14. 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.

  15. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Science.gov (United States)

    Sharon, Eilon; Shi, Hao; Kharbanda, Sandhya; Koh, Winston; Martin, Lance R; Khush, Kiran K; Valantine, Hannah; Pritchard, Jonathan K; De Vlaminck, Iwijn

    2017-08-01

    Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  16. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    2017-08-01

    Full Text Available Quantification of cell-free DNA (cfDNA in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD quantifies donor-derived cfDNA (dd-cfDNA by taking advantage of single-nucleotide polymorphisms (SNPs distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM of identity-by-descent (IBD states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD. These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

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

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2016-01-01

    Full Text Available Background. Noninvasive prenatal screening (NIPS is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

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

    Directory of Open Access Journals (Sweden)

    Goli eSamimi

    2015-04-01

    Full Text Available A range of molecular alterations found in tumor cells, such as DNA mutations and methylation changes, is also reflected in cell-free circulating DNA (circDNA released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. DNA methylation is a common molecular alteration found in many cancer types. Unlike DNA mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. DNA methylation is reflected within circDNA and therefore detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

  19. Some important advances in DNA repair study on the mammalian cells

    International Nuclear Information System (INIS)

    Xia Shouxuan.

    1991-01-01

    In the recent years the study of DNA damage and repair in the mammalian cells has gone deeply at gene level and got the following advances: (1) For a long time DNA has been considered to be an uniform unit in case of damage and repair. Now this concept should be replaced by the non-random distribution of damage and heterogenous repair in the genome. These would allow us to study cellular mutagenesis, carcinogenesis, aging and dying processes in great detail, and would be beneficial to the elucidation of mechanisms of radiation sickness and chemical toxicology. (2) The advent of new techniques in molecular biology has made it possible to isolate and clone the human DNA repair genes. Up to now more than ten human DNA repair genes have been cloned and these works would have an important impact on the theoretical and practical study in this field. Because DNA repair system is very complicate, voluminous work should be done in the future. (3) The technique of gene transfer has been efficiently used in the study of DNA repair in mammalian cells and has made great contribution in the cellular engineering. It could modify the genetic behavior of the gene-accepting cells, and enhance the DNA repair ability to physical and chemical damages. Human gene therapy for DNA deficient diseases is now on the day

  20. Quantitative detection of residual E. coli host cell DNA by real-time PCR.

    Science.gov (United States)

    Lee, Dong Hyuck; Bae, Jung Eun; Lee, Jung Hee; Shin, Jeong Sup; Kim, In Seop

    2010-10-01

    E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.

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

    International Nuclear Information System (INIS)

    Wilcoxson, L.T.; Griffiths, T.D.

    1984-01-01

    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

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

    International Nuclear Information System (INIS)

    Shen, Yan Nan; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun-Ran; Kim, Su-Hyeon; Hwang, Sang-Gu; Park, Sang Jun; Kim, Chun-Ho; Lee, Kee-Ho

    2014-01-01

    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

  3. Effect of superposed electromagnetic noise on DNA damage of lens epithelial cells induced by microwave radiation.

    Science.gov (United States)

    Yao, Ke; Wu, Wei; Yu, Yibo; Zeng, Qunli; He, Jiliang; Lu, Deqiang; Wang, Kaijun

    2008-05-01

    To investigate the influence of the 1.8-GHz radiofrequency fields (RFs) of the Global System for Mobile Communications on DNA damage, intracellular reactive oxygen species (ROS) formation, cell cycle, and apoptosis in human lens epithelial cells (hLECs) and whether the effects induced by RF could be blocked by superposing of electromagnetic noise. After 24-hour intermittent exposure at the specific absorption rate of 1 W/kg, 2 W/kg, 3 W/kg, and 4 W/kg, the DNA damage of hLECs was examined by alkaline comet assay and immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (gammaH2AX) foci, respectively. ROS production was quantified by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell cycle and cell apoptosis were determined by flow cytometry. DNA damage examined by alkaline comet assay was significantly increased after 3 W/kg and 4 W/kg radiation (P radiation (P microwave radiation and sham exposure groups (P > 0.05). All the effects mentioned were blocked when the RF was superposed with 2 muT electromagnetic noise. Microwave radiation induced hLEC DNA damage after G(0)/G(1) arrest does not lead to cell apoptosis. The increased ROS observed may be associated with DNA damage. Superposed electromagnetic noise blocks microwave radiation-induced DNA damage, ROS formation, and cell cycle arrest.

  4. Unique cell-type specific patterns of DNA methylation in the root meristem

    Science.gov (United States)

    Kawakatsu, Taiji; Stuart, Tim; Valdes, Manuel; Breakfield, Natalie; Schmitz, Robert J.; Nery, Joseph R.; Urich, Mark A.; Han, Xinwei; Lister, Ryan; Benfey, Philip N.; Ecker, Joseph R.

    2016-01-01

    DNA methylation is an epigenetic modification that differs between plant organs and tissues, but the extent of variation between cell types is not known. Here, we report single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and small RNA transcriptomes for six cell populations covering the major cell types of the Arabidopsis root meristem. We identify widespread cell type specific patterns of DNA methylation, especially in the CHH sequence context. The genome of the columella root cap is the most highly methylated Arabidopsis cell characterized to date. It is hypermethylated within transposable elements, accompanied by increased abundance of transcripts encoding RNA-directed DNA methylation (RdDM) pathway components and 24 nt small RNAs. Absence of the nucleosome remodeler DECREASED DNA METHYLATION 1, required for maintenance of DNA methylation, and low abundance of histone transcripts involved in heterochromatin formation suggests a loss of heterochromatin may occur in the columella, thus allowing access of RdDM factors to the whole genome, and producing excess 24 nt small RNAs in this tissue. Together, these maps provide new insights into the epigenomic diversity that exists between distinct plant somatic cell types. PMID:27243651

  5. DNA replication kinetics in x-irradiated Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Gerner, E.W.

    1977-08-01

    The kinetics of semiconservative DNA replication have been studied in both asynchronous and synchronized Chinese hamster ovary cells (CHO) irradiated with x-ray doses up to 3000 rad. Amounts of DNA replicated were determined by isopycnic gradient centrifugation of DNA from cells which were incubated after irradiation in medium containing 5-bromodeoxyuridine (50 ..mu..g/ml) and 5-fluorodeoxyuridine (0.1 ..mu..g/ml). The results confirm that cells irradiated in early G/sub 1/ phase experience a delay in their entry into S phase. This G/sub 1/ block is dose independent in the range from 300 to 3000 rad and is 0.5 to 0.7 hr in length. Cells at the G/sub 1//S boundary are insensitive to x-ray induced perturbations of bulk DNA synthetic rates when exposed to doses less than 1000 rad. At doses in excess of 1000 rad, these cells are inhibited from replicating their DNA for a time, but ultimately replicate near-control levels of their DNA. Cells irradiated in S phase again show no effects of x-ray doses below 1000 rad on their ability to replicate bulk DNA. After a 3000-rad exposure, however, the rate of DNA replication in these S-phase cells is markedly reduced compared to that of controls. Irradiation of asynchronous cells with doses from 150 to 3000 rad does reduce the rate of semiconservative DNA replication in these cultures in a dose-dependent manner. These results confirm that x-ray doses greater than 1000 rad reduce the rate of DNA synthesis in irradiated S-phase cells, thus prolonging the length of S phase. The combined data from asynchronous or synchronized cultures, irradiated with x-ray doses less than 1000 rad, indicate that at least a portion of the reduction in DNA replication rates in irradiated asynchronous CHO cultures is due to the x-ray induced G/sub 1/ block, which reduces the overall number of cells in S phase after irradiation.

  6. Radiosensitive xrs-5 and parental CHO cells show identical DNA neutral filter elution dose-response: implications for a relationship between cell radiosensitivity and induction of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Iliakis, George; Okayasu, Ryuichi; Seaner, Robert

    1988-01-01

    The purpose of this work was to investigate a possible correlation between DNA elution dose-response and cell radiosensitivity. For this purpose neutral (pH 9.6) DNA filter elution dose-response curves were measured with radiosensitive xrs-5 and the parental Chinese hamster ovary (CHO) cells in the logarithmic and plateau phase of growth. No difference was observed between the two cell types in the DNA elution dose-response curves either in logarithmic or plateau phase, despite the dramatic differences in cell radiosensitivity. This observation indicates that the shape of the DNA elution dose-response curve and the shape of the cell survival curve are not causally related. It is proposed that the shoulder observed in the DNA elution dose-response curve reflects either partial release of DNA from chromatin, or cell cycle-specific alterations in the physicochemical properties of the DNA. (author)

  7. 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

    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......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...... 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...

  8. 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.

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

    International Nuclear Information System (INIS)

    Satoh, Katsuya; Wada, Seiichi; Narumi, Issay; Kikuchi, Masahiro; Funayama, Tomoo; Kobayashi, Yasuhiko

    2003-01-01

    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)

  10. Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

    Science.gov (United States)

    Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

    2014-05-01

    Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

  11. Cell-free DNA as a biomarker in stroke: Current status, problems and