Background: DNA strand breaks pose the greatest threat to genomic stability. Genetically determined mutagen sensitivity predisposes individuals to a variety of cancers, including glioma. However, polymorphisms in DNA strand break repair genes that may determine mutagen sensitivity are not well studied in cancer risk, especially in gliomas.
Methods: We correlated genotype data for tag single-nucleotide polymorphisms (tSNPs) of DNA strand break repair genes with a gamma-radiation-induced mutagen sensitivity phenotype [expressed as mean breaks per cell (B/C)] in samples from 426 glioma patients. We also conducted analysis to assess joint and haplotype effects of single-nucleotide polymorphisms (SNPs) on mutagen sensitivity. We further validate our results in an independent external control group totaling 662 subjects.
Results: Of the 392 tSNPs examined, we found that mutagen sensitivity was modified by one tSNP in the EME2 gene and six tSNPs in the RAD51L1 gene (P < 0.01). Among the six RAD51L1 SNPs tested in the validation set, one (RAD51L1 rs2180611) was significantly associated with mutagen sensitivity (P = 0.025). Moreover, we found a significant dose–response relationship between the mutagen sensitivity and the number of adverse tSNP genotypes. Furthermore, haplotype analysis revealed that RAD51L1 haplotypes F-A (zero adverse allele) and F-E (six adverse alleles) exhibited the lowest (0.42) and highest (0.93) mean B/C values, respectively. A similar dose–response relationship also existed between the mutagen sensitivity and the number of adverse haplotypes.
Conclusion: These results suggest that polymorphisms in and haplotypes of the RAD51L1 gene, which is involved in the double-strand break repair pathway, modulate gamma-radiation-induced mutagen sensitivity.
The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.
bleomycin; Comet assay; DNA diffusion assay; Eleutherodactylus johnstonei; 4-nitroquinoline-1-oxide
Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and γ-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls.
Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments.
These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.
Inter-individual variation in DNA repair capacity is thought to modulate breast cancer risk. The phenotypic mutagen sensitivity assay (MSA) measures DNA strand breaks in lymphocytes; women with familial and sporadic breast cancers have a higher mean number of breaks per cell (MBPC) then women without breast cancer. Here, we explore the relationships between the MSA and the Rad51 gene, which encodes a DNA repair enzyme that interacts with BRCA1 and BRCA2, in BRCA1 mutation carriers and women with sporadic breast cancer.
Peripheral blood lymphoblasts from women with known BRCA1 mutations underwent the MSA (n = 138 among 20 families). BRCA1 and Rad51 genotyping and sequencing were performed to identify SNPs and haplotypes associated with the MSA. Positive associations from the study in high-risk families were subsequently examined in a population-based case-control study of breast cancer (n = 1170 cases and 2115 controls).
Breast cancer diagnosis was significantly associated with the MSA among women from BRCA1 families (OR = 3.2 95%CI: 1.5-6.7; p = 0.004). The Rad51 5'UTR 135 C>G genotype (OR = 3.64; 95% CI: 1.38, 9.54; p = 0.02), one BRCA1 haplotype (p = 0.03) and in a polygenic model, the E1038G and Q356R BRCA1 SNPs were significantly associated with MBPC (p = 0.009 and 0.002, respectively). The Rad51 5'UTR 135C genotype was not associated with breast cancer risk in the population-based study.
Mutagen sensitivity might be a useful biomarker of penetrance among women with BRCA1 mutations because the MSA phenotype is partially explained by genetic variants in BRCA1 and Rad51.
Given the high incidence of breast cancer and that more than half of cases remain unexplained, the need to identify risk factors for breast cancer remains. Deficiencies in DNA repair capacity have been associated with cancer risk. The mutagen sensitivity assay (MSA), a phenotypic marker of DNA damage response and repair capacity, has been consistently shown to associate with the risk of tobacco-related cancers. Methods: In a case–control study of 164 women with breast cancer and 165 women without the disease, we investigated the association between mutagen sensitivity and risk of breast cancer using bleomycin as the mutagen. Results: High bleomycin sensitivity (>0.65 breaks per cell) was associated with an increased risk of breast cancer, with an adjusted odds ratio of 2.8 [95% confidence interval (CI) = 1.7–4.5]. Risk increased with greater number of bleomycin-induced chromosomal breaks (Ptrend = 0.01). The association between bleomycin sensitivity and breast cancer risk was greater for women who were black, premenopausal and ever smokers. Our data also suggest that bleomycin sensitivity may modulate the effect of tobacco smoking on breast cancer risk. Among women with hypersensitivity to bleomycin, ever smokers had a 1.6-fold increased risk of breast cancer (95% CI = 0.6–3.9, P for interaction between tobacco smoking and bleomycin sensitivity = 0.32). Conclusions: Increased bleomycin sensitivity is significantly associated with an increased risk of breast cancer in both pre- and postmenopausal women. Our observation that the effect of tobacco smoking on breast cancer risk may differ based on mutagen sensitivity status warrants further investigation.
We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2’-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct “sensitivity profile” that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB.
The mutagen sensitivity assay is an in vitro measure of DNA repair capacity used to evaluate intrinsic susceptibility for cancer. The high heritability of mutagen sensitivity to different mutagens validates the use of this phenotype to predict cancer susceptibility. However, genetic determinants of mutagen sensitivity have not been fully characterized. Recently, several studies found that three major cytosine DNA methyltransferases (DNMTs), especially DNMT1, have a direct role in the DNA damage response, independent of their methyltransferase activity. This study evaluated the hypothesis that sequence variants in DNMT1, DNMT3A and DNMT3B are associated with mutagen sensitivity induced by the tobacco carcinogen benzo[a]pyrene diol epoxide (BPDE) in 278 cancer-free smokers. Single-nucleotide polymorphisms (n = 134) dispersed over the entire gene and regulatory regions of these DNMTs were genotyped by the Illumina Golden Gate Assay. DNA sequence variation in the DNMT1 and DNMT3B loci was globally associated with breaks per cell (P < 0.04 for both). No global association between DNMT3A and breaks per cell was seen (P = 0.09). Two haplotypes in block1 of DNMT1 (H284) and 3B (H70) were associated with 16 and 24% increase in breaks per cell, respectively. Subjects with three or four adverse haplotypes of both DNMT1 and 3B had a 50% elevation in mean level of breaks per cell compared with persons without adverse alleles (P = 0.004). The association between sequence variants of DNMT1 and 3B and mutagen sensitivity induced by BPDE supports the involvement of these DNMTs in protecting the cell from DNA damage.
Evidence in the literature suggests that hyperthermia (HT) or inhibitors of calmodulin can increase the sensitivity of rodent cells to bleomycin (BLM) by interfering with DNA repair functions. In an attempt to explore methods of improving the efficacy of thermochemotherapy we have investigated the individual and combined effects of HT (44 degrees C) and the calmodulin inhibitor trifluoperazine (TFP, 30 micrograms ml-1) on early plateau phase monolayer cultures of mouse EMT6 tumour cells for simultaneous exposures to BLM. Early plateau phase cultures are relatively resistant both to HT and to BLM. The selected HT and TFP regimens (either alone or in combination) were non-toxic. Comparing the sensitizing effect (given by the ratio: Do BLM/Do BLM + modifier) of the various regimens on BLM-treated cells, we found that: TFP alone had a marginal effect (ratio 1.3), HT alone showed significant potentiation (ratio 19) and the combination of HT and TFP strongly sensitized (ratio greater than 110) cells to BLM cytotoxicity. We propose that the use of calmodulin inhibitors in thermochemotherapy is worthy of further evaluation.
OBJECTIVE—This study was designed to investigate DNA damage and/or repair capability, non-random chromatid breakage, and p53 expression in patients with colorectal cancer.
METHODS—The bleomycin sensitivity assay was used in a case-control study to compare the DNA damage repair system between colorectal cancer patients and controls. G-banding was used to search for non-random chromatid breaks. Immunocytochemistry was used to investigate p53 expression in tumour tissues and adjacent normal tissues.
RESULTS—It was found that cases typically had a higher number of chromosome breaks than controls (0.84 v 0.69 breaks/cell, p<0.01). After correction by sex and age, the difference was still significant (F=4.38, p<0.05). The correlation coefficient between mutagen sensitivity and age was 0.31(p<0.05) in controls and 0.18 (p>0.05) in cases. The ratio of odds ratios among bleomycin resistant, sensitive, and hypersensitive classes was 1:2.31:3.85. Overexpression of p53 was detected in 25 of 47 tumour tissues independent of tumour stage. Cases who had a family history of cancer were susceptible to the p53 aberration (p<0.05). Chromosomes 1p, 5q, and 14q were susceptible to breakage in patients with colorectal cancer.
CONCLUSION—Patients with colorectal cancer show increased bleomycin induced chromatid breaks and may have minor DNA repair deficiencies. p53 aberration is an early event in the development of colorectal cancer, but no definite correlation is found between p53 overexpression and mutagen sensitivity.
Keywords: bleomycin induced chromatid break; p53 tumour suppressor gene; colorectal cancer
Mutations altering BLM function are associated with highly elevated cancer susceptibility (Bloom syndrome). Thus, genetic variants of BLM and proteins that form complexes with BLM, such as TOP3A and RMI1, might affect cancer risk as well.
In this study we have studied 26 tagged single nucleotide polymorphisms (tagSNPs) in RMI1, TOP3A, and BLM and their associations with cancer risk in acute myeloid leukemia/myelodysplatic syndromes (AML/MDS; N = 152), malignant melanoma (N = 170), and bladder cancer (N = 61). Two population-based control groups were used (N = 119 and N = 156).
Based on consistency in effect estimates for the three cancer forms and similar allelic frequencies of the variant alleles in the control groups, two SNPs in TOP3A (rs1563634 and rs12945597) and two SNPs in BLM (rs401549 and rs2532105) were selected for analysis in breast cancer cases (N = 200) and a control group recruited from spouses of cancer patients (N = 131). The rs12945597 in TOP3A and rs2532105 in BLM showed increased risk for breast cancer. We then combined all cases (N = 584) and controls (N = 406) respectively and found significantly increased risk for variant carriers of rs1563634 A/G (AG carriers OR = 1.7 [95%CI 1.1–2.6], AA carriers OR = 1.8 [1.2–2.8]), rs12945597 G/A (GA carriers OR = 1.5 [1.1–1.9], AA carriers OR = 1.6 [1.0–2.5]), and rs2532105 C/T (CT+TT carriers OR = 1.8 [1.4–2.5]). Gene-gene interaction analysis suggested an additive effect of carrying more than one risk allele. For the variants of TOP3A, the risk increment was more pronounced for older carriers.
These results further support a role of low-penetrance genes involved in BLM-associated homologous recombination for cancer risk.
The sites of bleomycin-induced cleavage of purified and intracellular simian virus 40 DNA were examined. Breaks in purified DNA were mapped to several discrete sites that were distributed throughout the viral genome, but were not associated with a common genetic element. Double-stranded breaks were made in positions of the first single-stranded nick, and regions of cuts were unaffected by changes in DNA superhelicity. Bleomycin cut intracellular chromosomes at the same sites that were cleaved in purified DNA. These results indicate that SV40 DNA contains DNA secondary structures that are highly preferred sites for BLM cleavage. These conformations appear to be unaffected by nucleoproteins bound to DNA.
Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.
Bloom syndrome is caused by homozygous mutations in BLM, which encodes a RecQ DNA helicase. Patient-derived cells deficient in BLM helicase activity exhibit genetic instability—apparent cytogenetically as sister chromatid exchanges—and activated DNA damage signaling. In this report, we show that BLM-knockout colorectal cancer cells exhibited endogenous, ATM-dependent double-strand DNA break responses similar to those recently observed in Bloom syndrome patient-derived cells. Xenograft tumors established from BLM-deficient cancer cells were not radiosensitive, but exhibited growth impairment that was comparable to that of wild type tumors treated with a single, high dose of ionizing radiation. These results suggest that pharmacological inhibitors of BLM would have a radiomimetic effect, and that transient inhibition of BLM activity might be a viable strategy for anticancer therapy.
Bloom syndrome; BLM; ATM; DNA damage; cancer therapy; ionizing radiation; xenografts; gene targeting
Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIα (Top3α) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3α. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (γ-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3α, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with γ-H2AX but not with Top3α or PML. Thus, BLM appears to dissociate from Top3α and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in γ-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.
The cooperation between phorbol 12-myristate 13-acetate induced protein 1 (NOXA) and myeloid cell leukemia 1 (MCL1) is critical in the intrinsic apoptotic pathway. Human papillomavirus 16 (HPV16), by inducing p53 and pRb-E2F degradation, may play an essential role in development of squamous cell carcinoma of the head and neck (SCCHN) through NOXA-MCL1 axis-mediated apoptosis. Therefore, genetic variants of NOXA and MCL1 may modify the SCCHN risk associated with HPV16 seropositivity.
HPV16 serology was obtained by immunoadsorption assay. Four functional SNPs in the promoter of NOXA (rs9957673, rs4558496) and MCL1 (rs9803935, rs3738485) were genotyped for 380 cases and 335 frequency-matched cancer-free controls of non-Hispanic whites.
Associations between the four polymorphisms and SCCHN risk were not significant, while we observed a significantly joint effect on SCCHN risk between the polymorphisms and HPV16 seropositivity. Notably, this effect modification was particularly pronounced for oropharyngeal cancer in subgroups including never smokers, never drinkers and younger subjects.
Our results suggested that polymorphisms of NOXA and MCL1 may modify the risk of HPV16-associated oropharyngeal cancer. The further identification of population subgroups at higher risk provides evidence that HPV-targeting treatment may help benefit SCCHN. However, larger studies are needed to validate our findings.
NOXA; MCL1; HPV16; Genetic susceptibility; Squamous cell carcinoma of the head and neck
Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.
Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC) and without (Comet assay) DNA repair inhibitors (araC and hydroxyurea). Furthermore, acellular Comet assay (acellular assay) was performed to determine how single-strand breaks (SSBs) as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD), which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1) for all mutagens studied, LGDs were MN test ≦ Comet assay; (2) except for BLM, LGDs were Comet assay/araC ≦ MN test; (3) except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1) LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2) for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were exactly identical; (3) the power of the Comet assay to detect a low level of genotoxic potential can be elevated to a level higher than that of MN test by using DNA resynthesis inhibitors, such as araC and HU.
Fanconi anemia (FA) and Bloom's syndrome (BS) are rare hereditary chromosomal instability disorders. FA displays bone marrow failure, acute myeloid leukemia, and head and neck cancers, whereas BS is characterized by growth retardation, immunodeficiency, and a wide spectrum of cancers. The BLM gene mutated in BS encodes a DNA helicase that functions in a protein complex to suppress sister chromatid exchange. Of the fifteen FA genetic complementation groups implicated in interstrand cross-link repair, FANCJ encodes a DNA helicase involved in recombinational repair and replication stress response. Based on evidence that BLM and FANCJ interact, we put forward that crosstalk between BLM and FA pathways is more complex than previously thought. We propose testable models for how FANCJ and BLM coordinate to help cells deal with stalled replication forks or double strand breaks. Understanding how BLM and FANCJ cooperate will help to elucidate an important pathway to maintain genomic stability.
The Bloom protein (BLM) and Topoisomerase IIIα are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIα or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIα are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIα or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIα with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIα-Fanconi pathway that is critical for suppression of chromosome radial formation.
Bloom protein; Fanconi anemia; Genome stability; Sister chromatid exchange; Topoisomerase IIIα
Although tobacco and alcohol consumption are two common risk factors of head and neck cancer (HNC), other specific etiologic causes, such as viral infection and genetic susceptibility factors, remain to be understood. Human DNA is often damaged by numerous endogenous and exogenous mutagens or carcinogens, and genetic variants in interaction with environmental exposure to these agents may explain interindividual differences in HNC risk. Single nucleotide polymorphisms (SNPs) in genes involved in the DNA damage-repair response are reported to be risk factors for various cancer types, including HNC. Here, we reviewed epidemiological studies that have assessed the associations between HNC risk and SNPs in DNA repair genes involved in base-excision repair, nucleotide-excision repair, mismatch repair, double-strand break repair and direct reversion repair pathways. We found, however, that only a few SNPs in DNA repair genes were found to be associated with significantly increased or decreased risk of HNC, and, in most cases, the effects were moderate, depending upon locus-locus interactions among the risk SNPs in the pathways. We believe that, in the presence of exposure, additional pathway-based analyses of DNA repair genes derived from genome-wide association studies (GWASs) in HNC are needed.
DNA repair; molecular epidemiology; head and neck cancer; genetic variation; genome-wide association study
We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.
Saccharomyces cerevisiae; karyopherin; nuclear transport; single-stranded DNA binding protein; bleomycin
DNA repair by homologous recombination is one of the main processes of DNA double strand breaks repair. In the present work we performed a case-control study (304 cases and 319 controls) to check an association between the genotypes of the c.-61 G>T and the g.38922 C>G polymorphisms of the RAD51 gene and the g.96267 A>C and the g.85394 A>G polymorphisms of the BLM gene and breast cancer occurrence. Genotypes were determined in DNA from peripheral blood by PCR-RLFP and by PCR-CTPP. We observed an association between breast cancer occurrence and the T/G genotype (OR 4.41) of the c.-61 G>T-RAD51 polymorphism, the A/A genotype (OR 1.69) of the g.85394 A>G-BLM polymorphism and the A/A genotype (OR 2.49) of the g.96267 A>C-BLM polymorphism. Moreover, we demonstrated a correlation between intra- and intergenes genotypes combinations and breast cancer occurrence. We found a correlation between progesterone receptor expression and the T/G genotype (OR 0.57) of the c.-61 G>T- RAD51 polymorphism. We also found a correlation between the T/G genotype (OR 1.86) and the T/T genotype (OR 0.56) of the c.-61 G>T- RAD51 polymorphism and the lymph node metastasis. We showed an association between the A/A genotype (OR 2.45) and the A/C genotype (OR 0.41) of the g.96267 A>C-BLM polymorphism and G3 grade of tumor. Our results suggest that the variability of the RAD51 and BLM genes may play a role in breast cancer occurrence. This role may be underlined by a common interaction between these genes.
Breast cancer; Gene polymorphism; BLM gene; RAD51 gene; DNA double-strand breaks
Bloom’s syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3′–5′ DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.
The variant polymorphism in the gene MDM2, SNP309, leads to increased level of mdm2 protein and subsequent downregulation of p53 tumor suppressor pathway. Presence of this single nucleotide polymorphism (SNP) has been associated with earlier tumorigenesis in patients with Li-Fraumeni syndrome, as well as decreased survival in patients with CLL. In addition, cells homozygous (G/G) for SNP 309 were found to have ten fold increase resistance to topoisomerase II inhibitors in vitro.
We genotyped children (n=575) with de novo acute myeloid leukemia (AML) treated on three Children’s Oncology Group protocols (CCG 2941/2961/AAML 03P1) for the presence of SNP309. Healthy blood donors were genotyped as control population.
The variant G/G genotype was associated with an increased susceptibility to AML (OR 1.5; p=0.049). However, the presence of the variant allele at SNP309 did not modify disease response or toxicity in children treated on CCG protocols 2941/2961.
The variant SNP 309 influences susceptibility to pediatric AML, but does not impact overall response to therapy.
AML; MDM2; SNP 309; Children’s Oncology Group; susceptibility
There is accumulating evidence that an individual’s inability to accurately repair DNA damage in a timely fashion may in part dictate a predisposition to cancer. Dogs spontaneously develop lymphoproliferative diseases such as lymphoma, with the golden retriever (GR) breed being at especially high risk. Mechanisms underlying such breed susceptibility are largely unknown; however, studies of heritable cancer predisposition in dogs may be much more straightforward than similar studies in humans, owing to a high degree of inbreeding and more limited genetic heterogeneity. Here, we conducted a pilot study with 21 GR with lymphoma, 20 age-matched healthy GR and 20 age-matched healthy mixed-breed dogs (MBD) to evaluate DNA repair capability following exposure to either ionizing radiation (IR) or the chemical mutagen bleomycin. Inter-individual variation in DNA repair capacity was evaluated in stimulated canine lymphoctyes exposed in vitro utilizing the G2 chromosomal radiosensitivity assay to quantify clastogen-induced chromatid-type aberrations (gaps and breaks). Golden retrievers with lymphoma demonstrated elevated sensitivity to induction of chromosome damage following either challenge compared to either healthy GR or MBD at multiple doses and time points. Using the 75th percentile of chromatid breaks per 1,000 chromosomes in the MBD population at 4 hours post 1.0 Gy IR exposure as a benchmark to compare cases and controls, GR with lymphoma were more likely than healthy GR to be classified as “sensitive” (odds ratio = 21.2, 95% confidence interval 2.3-195.8). Furthermore, our preliminary findings imply individual (rather than breed) susceptibility, and suggest that deficiencies in heritable factors related to DNA repair capabilities may be involved in the development of canine lymphoma. These studies set the stage for larger confirmatory studies, as well as candidate-based approaches to probe specific genetic susceptibility factors.