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PubMed2002

Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes: role in mutagen sensitivity.

Mutagen sensitivity, measuring the extent of chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been associated with an increased risk of various human cancers. Sensitivity to bleomycin appears to have high heritability and is usually considered to reflect individual capacity to repair DNA lesions. Another potential contributor to variation in bleomycin sensitivity could be inherited differences in the metabolism of bleomycin. We assessed whether genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes (XMEs) could explain bleomycin sensitivity. Frequencies of bleomycin-induced chromatid breaks per cell (b/c) were determined for 80 healthy Caucasians. Genotypes of DNA repair genes XRCC (X-ray repair cross-complementing) 1 and 3 and XME genes bleomycin hydrolase (BLHX), glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) and N-acetyltransferase 2 (NAT2) were analyzed from leukocyte DNA using methods based on polymerase chain reaction. The mean number of chromatid b/c was increased in individuals with XRCC1 codon 280 variant allele (P = 0.002; two-sided Mann-Whitney test). Smokers carrying BLHX codon 1450 variant allele showed a decrease in the mean number of chromatid b/c (P = 0.036). In multiple linear regression models including adjustment for age, sex, smoking and genotype, the adjusted relative risks (and 95% confidence intervals) were 1.18 (0.98-1.41) and 0.84 (0.69-1.00) for carriers of XRCC1 codon 280 and BLHX codon 1450 variant alleles, respectively. XRCC1 codon 280 polymorphism had a significant effect (P = 0.012) in predetermining whether the individual was classified as non-sensitive, sensitive or hypersensitive to bleomycin. Although based on relatively few individuals, our results suggest that bleomycin sensitivity is partially explained by genetic polymorphisms affecting DNA repair (XRCC1) and in vitro metabolism of bleomycin (BLHX).

Arylamine N-Acetyltransferase↗

PubMed2001

Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes.

Individual susceptibility to carcinogens, an important determinant of disease risk, is influenced by host factors such as the ability to repair DNA lesions. In order to identify subjects who are at high risk, we have developed a microgel electrophoresis assay for use in molecular epidemiological studies. The assay was validated in a pilot case-control study: Peripheral blood lymphocytes were collected from 100 patients with lung cancer and 110 control patients without cancer and from the same hospital, and stored at -80 degrees C. After thawing, phytohaemagglutinin-stimulated cells were treated with bleomycin at 20 microg/ml for 30 min and the extent of DNA damage and DNA repair capacity were determined by microgel electrophoresis. Peripheral blood lymphocytes from patients with lung cancer were significantly more sensitive to mutagens than those from controls and showed reduced DNA repair capacity (both P < 0.001). Both endpoints were independent risk factors for smoking-related lung cancer. Repeated analysis of peripheral blood lymphocytes from the same individual demonstrated good reproducibility of the assay. Cryopreservation of the lymphocytes for less than or = 12 months did not significantly affect their sensitivity. Our standardized microgel electrophoresis assay is suitable for determining individual sensitivity to mutagens and DNA repair capacity: it is sensitive and faster than cytogenetic assays, and can be applied to native and cryopreserved peripheral blood lymphocytes.

Adult↗

PubMed1997

Direct correlation between DNA repair capacity and metastatic potential of K-1735 murine melanoma cells.

The purpose of this study was to determine whether the ability of K-1735 murine melanoma cells to repair DNA damage correlates with their metastatic potential. Three nonmetastatic clones, four metastatic clones, and three somatic-cell hybrids between metastatic and nonmetastatic clones were exposed to incident ultraviolet (UV) light (254 nm). Cell survival was determined by the microculture tetrazolium assay, which measures cell metabolic activity. DNA repair capacity was determined with a host-cell reactivation assay, which measures the activities of chloramphenicol acetyltransferase encoded by the reporter gene in both UV-damaged and undamaged plasmid (control) pCMV cat 40 h after transfection. No discernible differences in transfection efficiencies were found between K-1735 clones with low and high metastatic potential or between cells transfected with UV-damaged and control plasmids. DNA repair capacity directly correlated with cell survival (p < 0.05) and with metastatic potential in the K-1735 clones and somatic cell hybrids (p < 0.05). These data suggest that the intrinsic resistance of melanoma metastases to systemic chemotherapy may be due, in part, to the cells' enhanced DNA repair capacity.

Animals↗

PubMed1995

DNA repair synthesis and DNA fragmentation in primary cultures of human and rat hepatocytes exposed to cyproterone acetate.

Cyproterone acetate (CPA), a synthetic steroid used in human therapy, has recently been shown to cause DNA damage in cultured rat hepatocytes and in rat liver. In the present study we have investigated whether CPA also induces genotoxic effects in human hepatocytes. Genotoxicity of CPA was determined by measuring the capability of the compound for inducing DNA repair synthesis and for causing the formation of DNA single-strand breaks. Autoradiography and alkaline elution were used to quantitate DNA repair and DNA fragmentation, respectively. Exposure of hepatocytes to CPA for 20 h induced DNA repair synthesis in two hepatocyte preparations derived from males and in four of the five preparations derived from females. In cultures from some donors, induction of repair was detected at 1 microM CPA, the lowest concentration tested. The maximum effect generally occurred at 10-20 microM. Only a very slight increase in the frequency of DNA single-strand breaks was found following exposure of the hepatocytes to 50 microM CPA for 20 h. For comparative purposes, the effects of CPA on DNA repair and DNA fragmentation were also determined in cultured rat hepatocytes. A strong induction of DNA repair synthesis, but only a slight enhancement in DNA fragmentation was observed in CPA-treated hepatocytes derived from female rats. These results indicate that the measurement of repair is a more sensitive indicator for the genotoxicity of CPA than the measurement of DNA fragmentation. No genotoxic effects of CPA were detectable in hepatocyte cultures derived from male rats. The present findings show that CPA is genotoxic in human hepatocytes and that the striking sex difference in the genotoxicity of CPA in rat cells is not observed with human cells.

Aged↗

PubMed2001

Error-prone repair DNA polymerases in prokaryotes and eukaryotes.

DNA repair is crucial to the well-being of all organisms from unicellular life forms to humans. A rich tapestry of mechanistic studies on DNA repair has emerged thanks to the recent discovery of Y-family DNA polymerases. Many Y-family members carry out aberrant DNA synthesis-poor replication accuracy, the favored formation of non-Watson-Crick base pairs, efficient mismatch extension, and most importantly, an ability to replicate through DNA damage. This review is devoted primarily to a discussion of Y-family polymerase members that exhibit error-prone behavior. Roles for these remarkable enzymes occur in widely disparate DNA repair pathways, such as UV-induced mutagenesis, adaptive mutation, avoidance of skin cancer, and induction of somatic cell hypermutation of immunoglobulin genes. Individual polymerases engaged in multiple repair pathways pose challenging questions about their roles in targeting and trafficking. Macromolecular assemblies of replication-repair "factories" could enable a cell to handle the complex logistics governing the rapid migration and exchange of polymerases.

Animals↗

PubMed1981

Induction of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase in regenerating rat liver.

The capacity of eukaryotic cells to modulate the activities of DNA repair enzymes during cell proliferation was examined. Using regenerating rat liver as a model system, the specific activities of the DNA repair enzymes uracil DNA glycosylase and 3-methyladenine DNA glycosylase were determined at specific intervals after partial hepatectomy. The induction of DNA replication and the stimulation of DNA polymerase were also measured in order to relate changes in the potential for DNA repair to those observed for DNA replication. As measured in nuclear extracts, the specific activities of both the uracil DNA glycosylase and the 3-methyladenine DNA glycosylase were increased in regenerating rat liver reaching maximal levels 18--24 h after partial hepatectomy. The specific activity of each DNA repair enzyme returned to basal levels by 48 h after the hepatectomy. No increase in either enzyme activity was observed in sham operated controls. The products of the reactions were identified as 3-methyladenine or as uracil by high pressure liquid chromatography or by gel filtration on Sephadex G-10. The 2--3 fold increases in the specific activity observed for each nuclear DNA repair enzyme was comparable to the 2.7 fold increase observed for DNA polymerase activity. The stimulation of DNA repair enzymes in regenerating rat liver is a further suggestion that eukaryotic cells actively regulate excision repair pathways in the defined pattern of gene expression observed during the eukaryotic cell cycle.

Animals↗

PubMed1990

[The regulation of DNA repair in mammalian cells. I. The repair of DNA radiation damages in Swiss 3T6 mouse cells under the action of the epidermal growth factor and insulin].

Methods of centrifugation in alkaline sucrose gradients as well as alkaline and neutral elution on filters were used to show a significant reduction in the rate of both single- and double-strand DNA breaks in the quiescent mouse Swiss 3T6 cell culture as compared to the proliferating one. The low efficiency of repair of single-strand DNA breaks in quiescent cells may result from a nearly complete absence of the fast repair of DNA lesions during the first minutes of postradiation incubation. The epidermal growth factor in combination with insulin (no other serum component present) leads to a recovery of the repair process. The stimulating effect of mitogens on the repair of both single- and double-strand DNA breaks allows to suggest that similar factors may be responsible for these recovery processes.

Animals↗

PubMed1989

[Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells].

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.

4-Nitroquinoline-1-oxide↗

PubMed1992

Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome.

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.

Adenosine Triphosphatases↗

PubMed2001

BCR-ABL down-regulates the DNA repair protein DNA-PKcs.

This study demonstrates in both stable and inducible BCR-ABL-expressing hematopoietic cells a down-regulation of the major mammalian DNA repair protein DNA-PKcs by BCR-ABL. Similar results were found in BCR-ABL CD34(+) cells from patients with chronic myelogenous leukemia (CML). DNA-PKcs down-regulation is a proteasome-dependent degradation that requires tyrosine kinase activity and is associated with a marked DNA repair deficiency along with increased sensitivity to ionizing radiation. The conjunction of a major DNA repair deficiency and a resistance to apoptosis, both induced by BCR-ABL, provides a new mechanism to explain how secondary genetic alterations can accumulate in CML, eventually leading to blast crisis. The down-regulation of DNA-PKcs was reversible in CD34(+) CML cells suggesting that this approach might offer a novel and powerful therapeutic strategy in this disease, especially to delay the blast crisis. (Blood. 2001;97:2084-2090)

Acetylcysteine↗

PubMed1984

Quantitative predictivity of carcinogenicity for four short-term parameters, evaluated in rat liver: alkaline DNA fragmentation, autoradiographic repair, DNA adducts, preneoplastic nodules.

The possibility of the study of a quantitative correlation between short-term tests and carcinogenicity, instead of a qualitative one, is discussed. Four tests related to the target organ, rat liver, were considered: alkaline DNA fragmentation, DNA repair, DNA adducts and the formation of preneoplastic nodules. All the four tests showed a similar level of correlation with carcinogenic potency (r approximately equal to 0.4). With this level of correlation, the dispersion of the data appeared too large to offer a meaningful degree of quantitative predictivity of carcinogenicity, in reference to a single test. It appeared however, that the use of a battery of two or three independent short-term tests, with the above level of simple correlation, could generate a multiple correlation high enough to be potentially useful for some degree of quantitative predictivity of carcinogenic potency.

Amines↗

PubMed2002

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes.

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

Cell Line, Transformed↗

PubMed2004

Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA.

We analysed the associations between genetic polymorphisms in genes coding for DNA repair enzymes XPD (exon 23 A --> C, K751Q), XPG (exon 15 G --> C, D1104H), XPC (exon 15 A --> C, K939Q), XRCC1 (exon 10 G --> A, R399Q) and XRCC3 (exon 7 C --> T, T241 M) and the levels of chromosomal aberrations (CAs) and single-strand breaks (SSBs) in peripheral lymphocytes in a central European population. We also measured the irradiation-specific DNA repair rates and the repair rates of 8-oxoguanines in these individuals. An elevated frequency of CAs was observed in individuals with the XPD exon 23 A allele (AA and AC) genotypes (F = 3.6, P = 0.028, ANOVA). In multifactorial analysis of variance, the XPD exon 23 polymorphism appeared as a major factor influencing CAs (F = 4.2, P = 0.017). SSBs in DNA, on the other hand, were modulated by XPD (F = 4.3, P = 0.023), XPG (F = 4.3, P = 0.024) and XRCC1 genotypes (F = 3.0, P = 0.064). Irradiation-specific DNA repair rates (reflecting mainly base excision repair activity) were affected by XRCC1 (F = 5.9, P = 0.010) and XPC polymorphisms (F = 4.2, P = 0.046, MANOVA). Our results from this study suggest that markers of genotoxicity are associated with polymorphisms in genes encoding DNA repair enzymes.

Adult↗

PubMed1978

Ultrastructural study of the nuclei in premitotic and repair DNA synthesis following UVB injury.

Ultrastructural changes in nuclei synthesizing DNA were studied by cytochemical technique. Guninea pigs ears were UVB irradiated and TdR-H3 was injected intradermally into the irradiated sites 1 hr before biopsy. Areas of the epidermis containing more than 80% of cells in DNA (repair or premitotic) synthesis identified by light microscopic autoradiography were selected and cut at 600 A. The glycolmethacrylate sections were stained with uranyl acetate and lead citrate, and consecutive sections were incubated with 0.01% pronase and 0.5% RNase before staining in order to observe DNA. In cells undergoing DNA repair, the zone of DNA became discontinuous and DNA was scattered throughout the entire karyoplasm as small aggregates and fine filaments. Nuclei in S-phase showed essentially the same change, but quantitatively the disappearance of DNA from the nuclear membrane and distribution in the karyoplasm became much greater. These changes were not seen in specimens treated without cytochemical technique.

Animals↗

PubMed1998

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication.

The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA. Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication. The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents. The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant. The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1. RAD2, a homolog of RTH1, is required for nucleotide-excision repair. Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7. Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments. Our data also indicate that only RTH1 functions in the repair of alkylation damage. Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene. These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair. The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

Alkylation↗

PubMed1996

Molecular epidemiology of skin cancers: DNA repair and non-melanocytic skin cancer.

Exposure to sunlight has been clearly associated with non-melanocytic skin cancer, however individual susceptibility differs considerably. Ethnic origin and pigmentary characteristics are generally accepted as risk factors. Another important defense mechanism is the ability to repair DNA photoproducts. In the inherited disorder xeroderma pigmentosum the inability to repair such DNA damage, associated with a high incidence of skin cancers, suggests an important role for DNA repair capacity in the etiology of this cancer. The development of an in vitro repair assay based on human peripheral blood lymphocytes, has allowed the evaluation of DNA repair capacity as a risk factor in population based studies. This article reviews recent developments in this field.

Animals↗

PubMed2003

Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth.

To evaluate the genetic control of stress responses in Arabidopsis, we have analyzed a mutant (uvh6-1) that exhibits increased sensitivity to UV light, a yellow-green leaf coloration, and mild growth defects. We have mapped the uvh6-1 locus to chromosome I and have identified a candidate gene, AtXPD, within the corresponding region. This gene shows sequence similarity to the human (Homo sapiens) XPD and yeast (Saccharomyces cerevisiae) RAD3 genes required for nucleotide excision repair. We propose that UVH6 is equivalent to AtXPD because uvh6-1 mutants carry a mutation in a conserved residue of AtXPD and because transformation of uvh6-1 mutants with wild-type AtXPD DNA suppresses both UV sensitivity and other defective phenotypes. Furthermore, the UVH6/AtXPD protein appears to play a role in repair of UV photoproducts because the uvh6-1 mutant exhibits a moderate defect in the excision of UV photoproducts. This defect is also suppressed by transformation with UVH6/AtXPD DNA. We have further identified a T-DNA insertion in the UVH6/AtXPD gene (uvh6-2). Plants carrying homozygous insertions were not detected in analyses of progeny from plants heterozygous for the insertion. Thus, homozygous insertions appear to be lethal. We conclude that the UVH6/AtXPD gene is required for UV resistance and is an essential gene in Arabidopsis.

Adenosine Triphosphatases↗

PubMed1998

The effect of nicotine on DNA repair in adult myocytes.

Recent findings have demonstrated that terminally differentiated adult ventricular myocytes are capable of repairing DNA that has been damaged by exposure to oxygen free radicals. Despite the potential importance of DNA repair in cells that may survive many decades after injury, little is known about the mechanisms or regulation of repair. Since tobacco use has a well-defined role in the epidemiology and pathophysiology of heart disease, we tested the effects of nicotine on repair of free radical damaged plasmids by whole-cell protein extracts from adult myocytes. Exposure to a concentration of 25 microM nicotine increased incorporation of (32P)dCTP into damaged plasmids by 16%, and 50 or 100 microM nicotine increased incorporation by 32%. Nicotine did not alter the rate or amount of poly (ADP-ribose) on the major protein acceptor of molecular weight 113-116 kDa. Inhibition of DNA polymerase activity with pyridoxal 5'-phosphate revealed greater plasmid degradation in the presence of nicotine. We conclude that nicotine enhances DNA degradation and the increased repair is a consequence of this greater degradation.

Animals↗