Low DNA Repair Capacity Research Articles

557 POLYMORPHISMS IN DNA REPAIR GENES MODIFY SURVIVAL OF BLADDER CANCER PATIENTS

INTRODUCTION AND OBJECTIVES: A role of genetic variability in DNA repair genes in response to therapies and survival has been reported for lung cancer but not for bladder cancer. Aim of the present study is to investigate the association between 28 SNPs in 8 different DNA repair genes and survival from bladder cancer. METHODS: The study population included all the newly diagnosed, histologically confirmed cases (456 patients) of bladder cancers registered at one single hospital during the years 19942008. All subjects were men, aged 40-75 years and living in the Turin metropolitan area. Before any treatment, a trained interviewer used a detailed questionnaire to conduct a face-to-face interview with patients on their history of tobacco smoking, occupational history, a 22-item food frequency questionnaire and a 24-h medication use recall. The type of therapy (BCG, chemotherapy, radiotherapy, etc) was recorded through the perusal of clinical records, in collaboration with urologists. We selected 28 SNPs in 8 different DNA repair genes because: 1) they have been associated with bladder cancer risk in previous studies and/or 2) they cause amino acidic substitution and/or 3) they are tagging SNPs. For each SNP we estimated the Hazard Ratios (HR) and 95% Confidence Interval (95% CI). We reconstructed haplotypes and imputed the phase by using a Bayesian method. RESULTS: 156 patients died during observation time. Median time of survival was 5.83 years. Variant alleles of five different polymorphisms in XRCC1 gene conferred a better survival, only among patients treated with chemotherapy (rs915927 adjHR 0.37 (95% CI 0.19-0.75); rs76507 adjHR 0.46 (95% CI 0.25-0.84); rs2854501 adjHR 0.34 (95% CI 0.15-0.73); rs2854509 adjHR 0.27 (95% CI 0.12-0.63); rs3213255 adjHR 0.44 (95% CI 0.24-0.82)). The score for the number of variant alleles in the XRCC1 gene showed a trend of increasing survival, only among patients treated with chemotherapy (29% decrease of risk of death for each additional variant allele in XRCC1; 0% in patients treated otherwise). Functional studies performed in a group of healthy subjects showed that the XRCC1 SNP variants associated to survival in this study conferred lower DNA repair capacity. CONCLUSIONS: Patients with a higher number of genetic variants in the XRCC1 DNA repair gene, who received chemotherapy, had a significantly reduced risk of death. A proficient DNA repair can result in cellular resistance to therapy and in worse survival. This finding may have clinical implications.

Modulation of DNA Repair Capacity by Ataxia Telangiectasia Mutated Gene Polymorphisms among Polycyclic Aromatic Hydrocarbons–Exposed Workers

The purpose of this study was to address the association between the ataxia telangiectasia mutated (ATM) gene polymorphisms and susceptibility to DNA repair capacity (DRC) among polycyclic aromatic hydrocarbons (PAHs)-exposed workers. Polymorphisms of ATM were genotyped. DRC was determined by comet assay. Chromosomal damage was detected by cytokinesis-block micronucleus (CBMN) assay. Flow cytometry was used to detect the distributions of cell cycle. Expressions of ATM and rH2AX were determined by immunoblotting analysis. Luciferase assays were performed to determine the functional difference of ATM promoter region allele. Subjects carrying T allele of rs228589 had significantly lower DRC compared with those with AA genotype. Subjects carrying G allele of rs652311 had significantly lower DRC than those with zero copy number of haplotype CGGT. SH ataxia telangiectasia mutated (SHATM) cells had significantly lower DRC than SH green fluorescent protein (SHGFP) cells induced by bleomycin and higher CBMN frequencies treated by benzo(a)pyrene [B(a)P] than SHGFP cells. After B(a)P treatment, a decrease in the percentage of G1 phase cells was observed in SHATM cells compared with SHGFP cells, rH2AX expressions were increased in SHATM cells and SHGFP cells, but ATM expressions had no change in 16HBE-SHGFP cells and HEK-SHGFP cells. Luciferase expression was not different between rs228589T and rs228589A plasmid constructs. In conclusions, it is suggested that ATM polymorphisms are associated with DRC among PAHs-exposed workers and ATM plays key roles in repair of chromosomal damage and cell cycle control with the treatment of B(a)P.

Abstract 1206: HER2/neu positive receptor status is associated with a low DNA repair capacity in women with breast cancer

Abstract Hormone receptor status are now routinely used for therapy selection in breast cancer (BC). Estrogen receptor status is routinely use for the selection of tamoxifen adjuvant therapy after surgery. Substantial evidence now indicates that patients with BC tumors that have overexpression of the HER2/neu receptor have generally a poor prognosis. Approximately 15-20 percent of BC patients have an amplification of the HER2/neu gene. Overexpression of HER2/neu is associated with increased BC recurrence and worse prognosis. Because of its prognostic role as well as its ability to predict response to trastuzumab (Herceptin), breast tumors are routinely checked for overexpression of HER2/neu. Strategies targeting DNA repair defects for the treatment of patients with triple-negative and BRCA mutation-associated BC have received much attention recently, however the relationship between HER2/neu receptor status and the phenotypic expression of DNA repair capacity (DRC) measured in lymphocytes has never been previously examined. DRC is an important risk factor for several types of cancer including BC. In this study, we examined the association between BC and DRC and the status of estrogen, progesterone and HER2/neu receptors. Our main objective was to determine if levels of DRC measured in lymphocytes using a host cell reactivation assay with a luciferase reporter gene are associated with receptor status. The estrogen, progesterone and HER2/neu receptor status, DRC levels, and other selected covariates were compared among 125 Hispanic women with histopathologically confirmed BC. After variable transformation, the mean DRC was compared between positive and negative HER2/neu BC patients using the t-test and the Wilcoxon's test to assess the statistical significance of the mean DRC difference. Crude and multiple logistic regression adjusted odds ratios (OR) were used as measures of association when compared HER2/neu (+) with (−) BC patients. The two-sided Wald test was used to assess the adjusted OR statistical significance. BC women with a HER2/neu (+) (n=14) had 4.1 times more odds of having a low DRC (< 1.78) compared with those that were HER2/neu (−) (n=65): (OR = 4.3 (95% CI 1.1, 16.6) p = 0.047. The average DRC in controls without BC was 6.10% (n=560). Women with BC that were HER2/neu (+) had an average DRC of 1.6% while BC patients that were HER2/neu (−) had 2.4%, this difference was significant (p=0.008). No significant differences were evident in regards to the association between DRC and the status of estrogen and progesterone receptors (double negatives) nor with triple negative receptor status (estrogen, progesterone and HER2/neu).These results indicate that low DRC could, at least partially explain, why HER2/neu (+) tumors are more aggressive. Supported by the NCI Diversity Training Branch of the Center to Reduce Cancer Health Disparities through the MBRS SCORE Program (Grant SO6 GM008239-23). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1206. doi:10.1158/1538-7445.AM2011-1206

DNA repair capacity marks susceptibility in pediatric asthma patients following low levels of polycyclic aromatic hydrocarbon exposure

We studied DNA damage, DNA repair capacity and other oxidative stress markers with environmental polycyclic aromatic hydrocarbon (PAH) exposure to find susceptible groups to show significant correlation between PAH exposure and studied markers. The total of study subjects were fifty five children from 6 to 9 years old including thirty six control group and nineteen asthma patients. No significant correlation between DNA damage and exposure markers was found among control group and asthma patients based on total antioxidants activity and glutathione/oxidized glutathione (GSH/GSSG) ratio. However, significant correlation was found between Tail DNA and 2-naphthol in high residual DNA damage group (20 upper percentile group) (r=0.63, p=0.038) who showed low DNA repair capacity. These studies revealed that low DNA repair capacity in children may render this population susceptible to environmental exposure including DNA damage-causing agents.

Evidence of sex and gender differences spurs call for more research funding

Evidence of sex and gender differences spurs call for more research funding

Abstract 3469: Effect of age and mutagen on the enriched murine mammary epithelial stem and progenitor cell population

Abstract Epidemiological studies indicated that aging is a significant risk factor for breast cancer. Yet, there is insufficient knowledge on the mechanisms affecting breast cancer initiation and promotion in older women. Recent studies indicated epithelial stem/progenitor cell population, responsible for lifelong tissue maintenance and regeneration, as a target of transformation. It has been reported earlier by serial transplantation of murine mammary tissues that functional mammary outgrowths indicating stem cell function is unaffected by the age of the donor. But it is unclear that whether spontaneous or induced mutagenesis in the aging stem/progenitor cell population has the potential to impair stem/progenitor functions and/or to initiate tumorigenesis. In this study, first we have isolated lineage negative (CD31−CD45−Ter119−) mammary epithelial cells (Lin−MEC) from the thoracic/inguinal mammary glands of young (6 months, n=7) and old (28 months, n=6) mice by serial enzymatic dissociation followed by magnetic nanoparticle separation method (StemCell Technology). These cells were utilized to form mammospheres, representing mammary stem/early progenitor cells. Although the frequency of mammosphere formation remained the same, the number of larger mammospheres (>60µm) from old mice were significantly lower than the young mice, probably indicating decreased stem cell activity in the older population. To study the effect of mutagen in vivo, we treated two young and two old mice with methyl nitrosourea (MNU, 25mg/kg body weight) once a week for three consecutive weeks. Animals were sacrificed one week after the last treatment. Both total and larger mammospheres (>60µm) from the isolated Lin−MEC of the treated older mice were observed in significantly higher number than the treated younger mice. In a recent clinical study larger mammospheres (>50µm) obtained from metastatic breast cancer cells has been correlated to their ability to induce tumors. These Lin−MEC cells were also analyzed and compared for DNA damage by alkaline comet assay and a higher percentage of cells (>10%) with irreversible DNA damage in the older population were observed compared to younger population (<2%) indicating probable lower DNA repair capacity of the former cells. Thus, our preliminary study indicated that enriched stem/progenitor cells in the old mice with decreased in vitro stem cell activity might have the potential to be vulnerable to DNA damage by mutagens and subsequent tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3469.

Abstract A6: Ethnic differences in risk of lung cancer associated with repair capacity for tobacco carcinogen-induced DNA adduct

Abstract Lung cancer is initiated by tobacco carcinogens-induced DNA damage, such as DNA adducts caused by benzo[a]pyrene diol epoxide (BPDE). These adducts can be removed by an efficient nucleotide excision repair. We have previously reported that DNA repair capacity (DRC) measured by the host-cell reactivation assay is associated with risk of lung cancer in non-Hispanic whites. In the recent analysis with larger sample sizes including minorities, we found that the suboptimal DRC remains associated with increased risk of lung cancer in 1730 cases and 1683 controls of non-Hispanic whites. The mean values of DRC were 8.37% ± 2.8% (SD) in the cases vs. 9.04% ± 3.1% in the controls (P < 0.0001). The odds ratio (OR) was 1.42 (95% confidence interval [CI], 1.24 – 1.64) after adjustment for age, sex, pack-years of smoking, family history of cancer, and other assay-related covariates. The similar results and risk trend have been also found in 127 Mexican-American patients with lung cancer and 176 cancer-free controls with a mean DRC of 8.38% ± 2.7% in the cases vs. 9.04% ± 3.3% in the controls, and an adjusted OR of 1.67 (95% CI, 1.00 – 2.78), although the difference in mean DRC values was borderline significant (P = 0.056) because of the relatively small sample size. However, this difference in DRC for tobacco carcinogen-induced DNA adducts was not significant in 327 African-American patients with lung cancer and 325 cancer-free controls. The mean values of DRC were 8.90% ± 3.1% in the cases vs. 8.66% ± 2.8% in the controls (P = 0.301). The adjusted OR was 0.99 (95% CI, 0.70 – 1.39). The DRC level in African-American controls was non-significantly lower than other ethnic controls (P = 0.144); however, African-American patients had significantly higher DRC than non-Hispanic white patients and Mexican-Americans patients (P = 0.053). The results further confirmed that low DRC is associated with increased risk of lung cancer in non-Hispanic whites and also provided the evidence of this association in Mexican Americans. These findings help us understand the ethnic differences in the development of lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A6.

1096 Molecular signatures of disseminated tumour cells in metastatic breast cancer patients

Gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and cisplatin are commonly used in the treatment of many solid tumors, although the impact of chemotherapy is limited in metastatic non-small cell lung cancer. However, in clinical practice, there is a minority of patients who can attain long-term survival. Upregulation of mRNA transcripts has been linked to chemoresistance, and in some instances, mRNA expression has been correlated with polymorphisms. Cisplatin resistance is directly linked to the nucleotide excision repair system, specifically to the transcription-coupled nucleotide excision repair pathway that involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. Overexpression of ERCC1 correlates with poor survival in gemcitabine/cisplatin-treated non-small cell lung cancer patients. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; P = .03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biologic redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival and highlight the possibilities of individually tailored chemotherapy.

No Association between hOGG1, XRCC1, and XPD Polymorphisms and Risk of Reflux Esophagitis, Barrett's Esophagus, or Esophageal Adenocarcinoma: Results from the Factors Influencing the Barrett's Adenocarcinoma Relationship Case-Control Study

Reflux of gastric contents can lead to development of reflux esophagitis and Barrett's esophagus. Barrett's esophagus is a risk factor for esophageal adenocarcinoma. Damage to DNA may lead to carcinogenesis but is repaired through activation of pathways involving polymorphic enzymes, including human 8-oxoguanine glycosylase 1 (hOGG1), X-ray repair cross-complementing 1 (XRCC1), and xeroderma pigmentosum group D (XPD). Of the single nucleotide polymorphisms identified in these genes, hOGG1 Ser 326 Cys, XRCC1 Arg 399 Gln, and XPD Lys 751 Gln are particularly common in Caucasians and have been associated with lower DNA repair capacity. Small studies have reported associations with XPD Lys 751 Gln and esophageal adenocarcinoma. XRCC1 Arg 399 Gln has been linked to Barrett's esophagus and reflux esophagitis. In a population-based case-control study, we examined associations of the hOGG1 Ser 326 Cys, XRCC1 Arg 399 Gln, and XPD Lys 751 Gln polymorphisms with risk of esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis. Genomic DNA was extracted from blood samples collected from cases of esophageal adenocarcinoma (n = 210), Barrett's esophagus (n = 212), reflux esophagitis (n = 230), and normal population controls frequency matched for age and sex (n = 248). Polymorphisms were genotyped using TaqMan allelic discrimination assays. Odds ratios and 95% confidence intervals were obtained from logistic regression models adjusted for potential confounding factors. There were no statistically significant associations between these polymorphisms and risk of esophageal adenocarcinoma, Barrett's esophagus, or reflux esophagitis.

Repair Capacity for UV Light–Induced DNA Damage Associated with Risk of Nonmelanoma Skin Cancer and Tumor Progression

To examine the role of suboptimal DNA repair capacity (DRC) for UV light-induced DNA damage in the development of nonmelanoma skin cancer (NMSC) and tumor progression. We conducted a hospital-based case-control study of 255 patients with newly diagnosed NMSC [146 with basal cell carcinoma (BCC) and 109 with squamous cell carcinoma (SCC)] and 333 cancer-free controls. We collected information on demographic variables and risk factors from questionnaires, tumor characteristics from medical records, and lymphocytic DRC phenotype by the host-cell reactivation assay. Multivariable logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (95% CI). Overall, there was a relative 16% reduction in DRC in NMSC patients compared with controls (P < 0.001 for BCC and for SCC, respectively). DRC below the controls' median value was associated with increased risk significantly for BCC (OR, 1.62; 95% CI, 1.07-2.45) but borderline for SCC (OR, 1.63; 95% CI, 0.95-2.79) after adjustment for age, sex, and other assay-related covariates. When the highest tertile of controls' DRC was used as the reference, the intermediate and low DRC were associated with a statistically significant trend for increasing risk for both BCC (P(trend) = 0.007) and SCC (P(trend) = 0.020). However, patients with aggressive or multiple SCC tended to have a higher DRC than those with nonaggressive or single SCC. Reduced DRC is an independent risk factor for BCC and single or nonaggressive SCC but not for multiple primaries, local aggressiveness, or recurrence of NMSC.

Interindividual differences in initial DNA repair capacity when evaluating H2O2-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H(2)O(2))-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H(2)O(2), and there was a significant decrease in the H(2)O(2)-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H(2)O(2)-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H(2)O(2)-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.

A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.

XPA A23G, XPC Lys939Gln, XPD Lys751Gln and XPD Asp312Asn polymorphisms, interactions with smoking, alcohol and dietary factors, and risk of colorectal cancer

Polymorphisms in the XPD and the XPC gene have been associated with a lower DNA repair capacity. We determined the risk of colorectal cancer in association with the four polymorphisms XPA A23G, XPC Lys939Gln, XPD Lys751Gln and XPD Asp312Asn, and interactions between the polymorphisms and the environmental factors: smoking intensity, intake of alcohol, red meat, processed meat, fish and poultry, fruits and vegetables and dietary fibres, in relation to development of colorectal cancer in a study population of 405 colorectal cancer cases and a comparison group of 810 persons, nested within the Danish prospective cohort, Diet, Cancer and Health, of 57 053 cohort members. No association was found between the XPC Lys939Gln, XPA A23G, XPD Lys751Gln, and XPD Asp312Asn polymorphisms and risk of colorectal cancer. The association of the XPD Lys751Gln polymorphism was statistically significantly different between genders, with a lower risk of colorectal cancer among women carrying the variant allele. We observed a statistically significant interaction between the XPC Lys939Gln polymorphism and consumption of red meat, with a 3.7-fold increase in colorectal cancer risk per 100 g red meat intake per day among carriers of the homozygous variant, but virtually no effect of red meat intake among carriers of the wild type allele. In the light of the multiple comparisons being made, this result may be a chance finding. The results showed no interaction between the XPD Lys751Gln, XPA A23G, and XPD Asp312Asn polymorphisms and the environmental factors for the development of colorectal cancer. Overall, the results of the present study indicate that the four polymorphisms are not of major importance in colorectal cancer carcinogenesis.

Cutaneous photoprotection and melanoma susceptibility: reaching beyond melanin content to the frontiers oF DNA repair

Melanoma is the most devastating form of skin cancer. The steady increase in the incidence of melanoma, its resistance to chemotherapy, together with its high potential to metastasize, have emphasized the importance of its prevention. It is becoming clear that solar ultraviolet radiation is a main culprit in the etiology of melanoma, the same as in basal and squamous cell carcinomas. It is commonly accepted that skin pigmentation and melanin content are principal determinants of the susceptibility to melanoma and other sun-induced skin cancers. Although this is generally true, however, prediction of melanoma risk based solely on pigmentary phenotype is not always precise and fails to identify high-risk individuals with dark skin color. Other important risk factors need to be considered and better defined, particularly DNA repair capacity. Emerging studies have revealed the role of melanoma susceptibility genes in regulating DNA repair, and indicated that melanoma patients have a lower DNA repair capacity than the general population. As the response of human melanocytes to ultraviolet radiation is modulated by an array of paracrine factors, we have focused our investigation on the role of melanocortins and the melanocortin 1 receptor, as well as endothelin-1, in this response. We have discovered novel roles for melanocortins and endothelin-1 as survival factors that rescue human melanocytes from ultraviolet radiation-induced apoptosis, and importantly enhance repair of DNA photoproducts and reduce the release of hydrogen peroxide that can cause oxidative stress. Our findings, together with epidemiological data showing that loss-of-function mutations in the melanocortin-1 receptor gene increase the risk of melanoma, substantiate the role of DNA repair in melanoma genesis, and suggest that responsiveness to melanocortins and endothelin-1 is important for melanoma prevention.

ERCC1, XPD and RAI mRNA levels in lymphocytes are not associated with lung cancer risk in a prospective study of Danes

Low DNA-repair capacity has been associated with increased risk of several types of cancer. mRNA levels of the nucleotide excision repair genes ERCC1 and XPD have been shown to correlate with the DNA-repair capacity. Likewise, mRNA levels of several DNA-repair genes including ERCC1 have been shown to be lower in lymphocytes from patients with lung cancer and head and neck cancer compared with healthy persons. In these studies, the low DNA-repair gene expression levels could be either a risk factor for disease or a consequence of the same. In this nested case–cohort study, which to our knowledge, is the first prospective study of DNA-repair gene mRNA levels as predictors of lung cancer, we have investigated the occurrence of lung cancer in relation to the mRNA level of the two DNA-repair genes ERCC1 and XPD and the NF κB inhibitor RAI in blood samples prior to disease. Among 54,220 members of a Danish prospective cohort study, 265 lung cancer cases were identified and a sub-cohort comprising 272 individuals was used for comparison. The expression levels of the three adjacent genes were found to be highly inter-correlated, to be higher in women compared to men and to be lower in older individuals. The incidence rate ratios for lung cancer in association with one log-unit increase (natural logarithm) in mRNA levels were 1.12 (CI = 0.89–1.41) for ERCC1, 1.00 (CI = 0.83–1.21) for XPD and 1.25 (0.89–1.74) for RAI. In conclusion, this study indicated no association between mRNA expression of the DNA-repair genes ERCC1 and XPD and risk of subsequent development of lung cancer.

MC1R, ASIP, and DNA Repair in Sporadic and Familial Melanoma in a Mediterranean Population

Melanoma risk factors include fair pigmentation, multiple nevi, low DNA repair capacity, and CDKN2A or CDK4 mutations. Variants of the melanocortin-1 receptor (MC1R) gene have been associated with fair pigmentation and melanoma risk, and a polymorphism of the Agouti Signaling Protein (ASIP) gene has been associated with dark pigmentation. We examined MC1R and ASIP genotypes in relation to phenotypic characteristics, sporadic and familial melanoma risk, and melanoma thickness as an indicator of disease progression in a Mediterranean population. We studied 267 melanoma patients and 382 control subjects from a case-control study and a family study in northeastern Italy. Host factors were assessed by physical examination, questionnaire, spectrophotometer, and minimal erythema dose measurement. MC1R was sequenced, ASIP was genotyped, and DNA repair capacity was measured by the host-cell reactivation assay. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression models. Effect modification of the association between MC1R and melanoma risk by phenotypic characteristics and DNA repair capacity was also assessed. All statistical tests were two-sided. Carrying MC1R variant alleles was associated with a two- to fourfold increase in risk of both sporadic and familial melanoma compared with carrying wild-type MC1R, particularly in individuals carrying multiple variant alleles (OR = 3.9; 95% CI = 3.3 to 4.6). This association was stronger in individuals with fewer additional risk factors (those with dark skin or few nevi). MC1R variant allele carriers were also three to four times more likely than were non-carriers to have thick melanomas. The ASIP polymorphism was not associated with pigmentation, nevi, or melanoma risk. MC1R was associated with melanoma risk and progression in a Mediterranean population, particularly in the absence of other strong risk factors, such as freckling or many nevi.

Measuring DNA Repair Capacity: Small Steps

DNA repair capacity is a system of defenses designed to protect the integrity of the genome. The difficulty in studying DNA repair capacity in relationship to cancer etiology stems from the costs inherent in conducting whole cell assays, which represent an integrated response to DNA damage, and from the lack of specificity of the current genetic assays, which generally focus on the role of single nucleotide polymorphisms. Studies of DNA repair capacity and cancer etiology require careful consideration of design, potential bias and confounding, and, importantly, assessment of assay variability, both intra-individual and inter-individual, as well as biologic plausibility (1). Design issues center on the difficulty of selecting appropriate subjects for study. A major problem with almost all studies of DNA repair capacity and its role in cancer etiology is that susceptible individuals should be identified before the development of cancer, rather than after cancer has been diagnosed and treated. This is because the cancer itself and likely its treatment may result in observations slanted toward lower DNA repair capacity among case patients who may have been treated with radiation or chemicals. Therapies may modify DNA repair capacity, and, in fact, are designed to knock out the DNA repair capacity of the tumor. Potential biases and confounding in studies of DNA repair capacity can result from several factors: an inability to identify all relevant exposures; the inducibility of some DNA repair genes (1), which can occur as a result of exposure to many different agents; and those that represent biologic cross reactivity, age, dietary habits, and exposure to pro-oxidants (2). Measurement of assay variability is critical for assessing the true, underlying risk estimates. Because DNA repair capacity may be modified by environmental exposures—which could be useful as we consider the potential of DNA repair capacity to be a target for intervention (3) and a potential therapeutic target (4)—it is critical to understand the interand intra-individual variability in any particular set of assays. In this issue of the Journal, Kennedy et al. (5) report on the assay they developed for DNA repair capacity that takes advantage of the adducts induced by the carcinogenic metabolite of benzo[a]pyrene, benzo[a]pyrene diolepoxide (BPDE). BPDE adducts are repaired by the nucleotide excision repair pathway, which has been implicated along with the homologous recombination and the non-homologous end joining repair pathways in BRCA1/2 associated breast cancer. Kennedy et al. found that this measure of DNA repair capacity was lower in breast cancer case patients than control subjects. This study overcomes some previous limitations of case– control studies of breast cancer by using a robust study design, using a functional and specific characterization of nucleotide excision repair, BPDE, and controlling for daily assay variation using cell lines. Although some limitations are present, the findings should lead to renewed interest in DNA repair capacity as a risk factor for cancer and suggest new avenues for investigation. A major strength of the study is the family-based design using sisters from the same families that are discordant for breast cancer. The authors report that not only do younger breast cancer patients have statistically significantly lower DNA repair capacity than their sisters without breast cancer, but that also in multivariable models, breast cancer patients have a dose response increase in the odds of breast cancer with decreasing DNA repair capacity, ranging from odds of 1.23 (95% CI 0.57 to 2.65) to 2.38 (95% CI 1.17 to 4.86) to 2.99 (95% CI 1.45 to 6.17) (Ptrend .002). The potential confounder body mass index (BMI) was statistically significantly different in simple comparisons between case patients and control subjects. At a BMI equal to or greater than 25, indicative of overweight and obesity, case patients were statistically significantly more likely to have lower DNA repair capacity than control subjects. To our knowledge, this is the first evidence that overweight/obesity may affect DNA repair capacity. The critical issue of whether breast cancer patients have experienced therapy—either radiation or chemotherapy—has, unfortunately, not been mentioned or controlled for in this study. Radiation therapy, and possibly chemotherapy, appears to lead to decreased DNA repair capacity (6–8). Kennedy et al. (5) measured the daily variation in two cell lines, one DNA repair deficient and one DNA repair proficient. We would also be interested in the repeatability and variation of these assays on the individual lymphoblastoid cell lines over time. There is also the

Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B[a]PDE-DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers.

It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes [xeroderma pigmentosum-C (XPC)-PAT +/-, xeroderma pigmentosum-A (XPA) 5' non-coding region-A23G, XPD-exon 23 A35931C Lys751Gln, xeroderma pigmentosum-D (XPD)-exon 10 G23591A Asp312Asn] and that of glutathione S-transferase mu1 (GSTM1-active or -null) on benzo[a]pyrene diol epoxide (B[a]PDE)-DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo[a]pyrene (B[a]P)-exposed Polish coke oven workers (n = 67, 67% current smokers) with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index (BEI) (2.28 micromol/mol creatinine). The bulky (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE)-DNA adduct levels were detected by high-performance liquid chromatography (HPLC)/fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had significantly increased anti-B[a]PDE-DNA adduct levels (Mann-Whitney U-test, z = 2.24, P = 0.02 and z = 2.65, P = 0.01). Moreover, DNA adducts were also raised in workers without GSTM1 activity (GSTM1-null genotype) (Mann-Whitney U-test, z = 2.25, P = 0.0246). Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone (approximately 65% of workers) or combined with GSTM1-null genotype (approximately 75% of workers) were in the tertile with the highest adduct level, i.e. >4.11 adducts/10(8) nt (chi2 = 5.85, P = 0.0156 and chi2 = 5.40, P = 0.01). The increase in anti-B[a]PDE-DNA adduct levels (ln values) was significantly related in a multiple linear regression analysis to PAH exposure (i.e. urinary post-shift excretion of 1-pyrenol) (t = 2.61, P = 0.0115), lack of GSTM1 activity (t = 2.41, P = 0.0192) and to low DNA repair capacity of the XPC-PAT+/+ genotype (t = 2.34, P = 0.0226). The influence of the XPA-A23A genotype was not evident in this statistical analysis, and no associations with XPD polymorphisms, dietary habits or tobacco smoking were found. The modulation of anti-B[a]PDE-DNA adducts in the LMF by GSTM1-null and some low-activity NER genotypes may be considered as a potential genetic susceptibility factor capable of modulating individual responses to PAH (B[a]P) genotoxic exposure and the consequent risk of cancer in coke oven workers.

DNA repair and breast carcinoma susceptibility in women.

Breast carcinoma is the most common cancer and the second leading cause of cancer-related deaths among women. The disease represents approximately 31% of all cancers in Puerto Rican women. Several DNA repair pathways are involved in preventing carcinogenesis. The current study evaluated the hypothesis that a reduced DNA repair capacity (DRC) is a susceptibility factor for breast carcinoma. A retrospective case-control clinical study was performed to compare age-matched DRC in 33 women with histopathologically confirmed breast carcinoma (cases) and 47 cancer-free women (controls). DRC was measured using a host cell reactivation assay with a luciferase reporter gene and then transfected into human peripheral lymphocytes. A questionnaire was used to solicit breast carcinoma risk factors. Women with breast carcinoma had a mean DRC of 5.6% +/- 0.5 standard error of the mean (SEM). Cancer cases had a 36% reduction (P<0.001) in DRC when compared with the control group (DRC=8.7% +/- 0.7 SEM). Younger participants with breast carcinoma were found to have a more significant reduction in DRC when compared with age-matched controls. Family (odds ratio [OR]=4.1), maternal lineage (OR=5.5), and maternal (OR=12.4) history of breast carcinoma were found to be the only statistically significant (P<0.05) risk factors associated with the disease. The findings supported the hypothesis that a low DRC is a susceptibility factor for breast carcinoma. A 1% decrease in DRC corresponded to a 22% increase in breast carcinoma risk. To the authors' knowledge, the current study was the first to directly determine the DRC of women with breast carcinoma. Because DRC is an independent risk factor for breast carcinoma, the DRC of women may be a useful marker in predicting susceptibility.

XPD gene polymorphism and host characteristics in the association with cutaneous malignant melanoma risk.

We recently reported an association between low DNA repair capacity, measured through the host-cell reactivation assay, and melanoma risk in subjects with dysplastic naevi or low tanning ability. We investigated the genetic basis for these findings by analysing the Asp312Asn and Lys751Gln polymorphisms of the XPD (ERCC2) DNA repair gene in the same subjects. Similar to our previous report, no significant association between XPD polymorphisms and melanoma risk was found in 176 melanoma cases and 177 controls (odds ratio (OR)=1.5, 95% confidence interval (CI)=0.9–2.5 for 312Asn; OR=1.3, 95% CI=0.8–2.1 for 751Gln, adjusted for age, gender, dysplastic naevi and pigmentation characteristics). However, XPD variants were associated with increased risk in older (>50 years) subjects (OR=3.4, 95% CI=1.6–7.3 for 312Asn; OR=2.3, 95% CI=1.1–4.9 for 751Gln). The 751Gln allele was associated with elevated melanoma risk among subjects without dysplastic naevi (OR=2.6, 95% CI=1.1–6.4). Subjects with low tanning ability and XPD variants exhibited a nonsignificant increase of melanoma risk (OR=2.3, 95% CI=0.7–7.0 for 312Asn; OR=3.0, 95% CI=1.0–8.8 for 751Gln). DNA repair capacity was slightly decreased in subjects carrying 751Gln alleles. XPD variants may modify melanoma risk in subjects with specific host characteristics, such as older age, lack of dysplastic naevi or low tanning ability.