Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1+/-11.8) compared to whole blood (43.0+/-12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p=0.01) in ex-/non-smokers (41.0+/-2.1%) compared to smokers (50.3+/-2.7%). This phase of repair was also slower (p=0.02) in HNSCC patients (36.8+/-2.1%) compared to controls matched on age and smoking (46.4+/-3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.     

DNA damage among thyroid cancer and multiple cancer cases, controls, and long-lived individuals

Variation in the detection, signaling, and repair of DNA damage contributes to human cancer risk. To assess capacity to modulate endogenous DNA damage among radiologic technologists who had been diagnosed with breast cancer and another malignancy (breast-other, n=42), early-onset breast cancer (early-onset, age or=75% versus below the median, age-adjusted) was most consistently associated with the highest odds ratios in the breast-other, early-onset, and thyroid cancer groups (with risk increased 10-, 5- or 19-fold, respectively, with wide confidence intervals) and decreased risk among the hyper-normal group. For the other three comet measures, risk of breast-other was elevated approximately three-fold. Risk of early-onset breast cancer was mixed and risk of thyroid cancer ranged from null to a two-fold increase. The hyper-normal group showed decreased odds ratios for tail DNA and OTM, but not CDM. DNA damage, as estimated by all comet measures, was relatively unaffected by survival time, reproductive factors, and prior radiation treatment. We detected a continuum of endogenous DNA damage that was highest among cancer cases, less in controls, and suggestively lowest in hyper-normal individuals. Measuring this DNA damage phenotype may contribute to the identification of susceptible sub-groups. Our observations require replication in a prospective study with a large number of pre-diagnostic samples.     

Repair kinetics of gamma-ray induced DNA damage determined by the single cell gel electrophoresis assay in murine leukocytes in vivo

The repair kinetics of the gamma rays induced DNA damage was determined in murine peripheral blood leukocytes in vivo by the comet assay. Mice were exposed to 1.0 Gy of gamma rays in a 137Cs source and samples of peripheral blood were taken from their tails at different times. The repair was evaluated per mice in separate experiments by measuring the proportion of cells with tail (comets) in each sample. An average of nearly 80% of comets was obtained at the initial time after the exposure; 2 min later the frequency decreased to 45% and continued diminishing to 22% at 15 min. This evidences the presence of a rapid repair mechanism. For a period of 25 to 40 min after exposure there was a slight but consistent increase of comets from 22 to 38% followed by a second reduction, which could be due to a late repair process that causes strand breaks and then joined them. In summary our results indicated that this system seems to be appropriate for the study of the repair capacity of cells following exposure to ionizing radiation.     

In vitro effect of gliclazide on DNA damage and repair in patients with type 2 diabetes mellitus (T2DM)

Type 2 diabetes mellitus is associated with elevated level of oxidative stress, which is one of the most important factors responsible for the development of chronic complications of this disease. Moreover, it was shown that diabetic patients had increased level of oxidative DNA damage and decreased effectiveness of DNA repair. These changes may be associated with increased risk of cancer in T2DM patients, since DNA damage and DNA repair play a pivotal role in malignant transformation. It was found that gliclazide, an oral hypoglycemic drug with antioxidant properties, diminished DNA damage induced by free radicals. Therefore, the aim of the present study was to evaluate the in vitro impact of gliclazide on: (i) endogenous basal and oxidative DNA damage, (ii) DNA damage induced by hydrogen peroxide and (iii) the efficacy of DNA repair of such damage. DNA damage and DNA repair in peripheral blood lymphocytes of 30 T2DM patients and 30 non-diabetic individuals were evaluated by alkaline single cell electrophoresis (comet) assay. The extent of oxidative DNA damage was assessed by DNA repair enzymes: endonuclease III and formamidopyrimidine-DNA glycosylase. The endogenous basal and oxidative DNA damages were higher in lymphocytes of T2DM patients compared to non-diabetic subjects and gliclazide decreased the level of such damage. The drug significantly decreased the level of DNA damage induced by hydrogen peroxide in both groups. Gliclazide increased the effectiveness of DNA repair in lymphocytes of T2DM patients (93.4% (with gliclazide) vs 79.9% (without gliclazide); P< or =0.001) and non-diabetic subjects (95.1% (with gliclazide) vs 90.5% (without gliclazide); P< or =0.001). These results suggest that gliclazide may protect against the oxidative stress-related chronic diabetes complications, including cancer, by decreasing the level of DNA damage induced by reactive oxygen species.     

DNA damage in leukocytes of workers occupationally exposed to 1-bromopropane

1-bromopropane (1-BP; n-propyl bromide) (CAS No. 106-94-5) is an alternative to ozone-depleting chlorofluorocarbons that has a variety of potential applications as a degreasing agent for metals and electronics, and as a solvent vehicle for spray adhesives. Its isomer, 2-brompropane (2-BP; isopropyl bromide) (CAS No. 75-26-3) impairs antioxidant cellular defenses, enhances lipid peroxidation, and causes DNA damage in vitro. The present study had two aims. The first was to assess DNA damage in human leukocytes exposed in vitro to 1- or 2-BP. DNA damage was also assessed in peripheral leukocytes from workers with occupational exposure to 1-BP. In the latter assessment, start-of- and end-of-work week blood and urine samples were collected from 41 and 22 workers at two facilities where 1-BP was used as a solvent for spray adhesives in foam cushion fabrication. Exposure to 1-BP was assessed from personal-breathing zone samples collected for 1-3 days up to 8h per day for calculation of 8h time weighted average (TWA) 1-BP concentrations. Bromide (Br) was measured in blood and urine as a biomarker of exposure. Overall, 1-BP TWA concentrations ranged from 0.2 to 271 parts per million (ppm) at facility A, and from 4 to 27 ppm at facility B. The highest exposures were to workers classified as sprayers. 1-BP TWA concentrations were statistically significantly correlated with blood and urine Br concentrations. The comet assay was used to estimate DNA damage. In vitro, 1- or 2-BP induced a statistically significant increase in DNA damage at 1mM. In 1-BP exposed workers, start-of- and end-of-workweek comet endpoints were stratified based on job classification. There were no significant differences in DNA damage in leukocytes between workers classified as sprayers (high 1-BP exposure) and those classified as non-sprayers (low 1-BP exposure). At the facility with the high exposures, comparison of end-of-week values with start-of-week values using paired analysis revealed non-sprayers had significantly increased comet tail moments, and sprayers had significantly increased comet tail moment dispersion coefficients. A multivariate analysis included combining the data sets from both facilities, log transformation of 1-BP exposure indices, and the use of multiple linear regression models for each combination of DNA damage and exposure indices including exposure quartiles. The covariates were gender, age, smoking status, facility, and glutathione S-transferase M1 and T1 (GSTM1, GSTT1) polymorphisms. In the regression models, start-of-week comet tail moment in leukocytes was significantly associated with serum Br quartiles. End-of-week comet tail moment was significantly associated with 1-BP TWA quartiles, and serum Br quartiles. Gender, facility, and GSTM1 had a significant effect in one or more models. Additional associations were not identified from assessment of dispersion coefficients. In vitro and in vivo results provide limited evidence that 1-BP exposure may pose a small risk for increasing DNA damage.     

Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells

The effect of exposure to elevated temperatures (41-45 degrees C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45 degrees C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45 degrees C for 15 min slowed the rate of DNA repair to 0.17 of the control repair rate. The To for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45 degrees C, respectively. Stepdown-heating (45 degrees C for 15 min followed by repair at 41 degrees C) resulted in greater inhibition of DNA repair (0.11 of the control rate) than was observed after acute heating alone. Repair at 41 degrees C was observed to proceed in unheated cells at a faster rate than at 37 degrees C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45 degrees C indicated an activation energy of 140 kcal mol-1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.     

Residential sunlight exposure is associated with a decreased risk of prostate cancer

The possibility that exposure to sunlight reduces the risk of clinical prostate cancer has been strongly suggested by ecologic data. However, data on prostate cancer risk in relation to sunlight exposure in individuals are sparse. We analyzed data from the First National Health and Nutrition Examination Survey (NHANES I) Epidemiologic Follow-up Study in order to test the hypothesis that residential sunlight exposure reduces the risk of prostate cancer. We identified 153 men with incident prostate cancer from a cohort of 3414 white men who completed the baseline interview and dermatologic examination in 1971-1975 and were followed up to 1992. We used Cox proportional hazards modeling to estimate relative risks (RR) and 95% confidence intervals (CI) for measures of residential sunlight exposure, adjusting for age, family history of prostate cancer, and dietary intake of fat and calcium. Residence in the South at baseline (RR = 0.68, CI = 0.41-1.13), state of longest residence in the South (RR = 0.62, CI = 0.40-0.95), and high solar radiation in the state of birth (RR = 0.49, CI = 0.30-0.79) were associated with significant and substantial reductions in prostate cancer risk. These data support the hypothesis that sunlight exposure reduces the risk of prostate cancer and have important implications for prostate cancer prevention.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Oxidative DNA Damage in the Prostate May Predispose Men to a Higher Risk of Prostate Cancer

DNA damage has been associated with prostate cancer risk. Men who were referred for initial prostate biopsy for elevated prostate-specific antigen or abnormal digital rectal examination are often found with no cancer but have a higher risk of developing prostate cancer than the general population of men in their lifetime. In this study, we investigated whether DNA damage is one of the factors that predispose these men referred for prostate biopsies to a higher risk of prostate cancer. We found significantly elevated levels of 8-oxo-2-deoxyguanosine immunoreactivity in the prostates of the referred men (n = 50) in comparison to the control prostates of men (n = 32) with no indication for referral for prostate biopsy. Twelve of these control men were healthy middle-aged men and 20 of them were older men whose conditions were diagnosed with bladder cancer but with normal serum prostate-specific antigen and digital rectal examination and no evidence of prostate disease. In all the 8-oxo-2-deoxyguanosine-positive prostates, we detected phosphorylation of the ataxia telangiectasia mutated kinase and expression of the immune-stimulatory molecule MIC in the prostate epithelium. These data suggest that: 1) oxidative DNA damage has occurred in the “referred” but pathologically normal prostates, indicating that these prostates may be subjected to genomic instability and eventually neoplastic transformation; 2) in response to DNA damage, two surveillance pathways, represented by ataxia telangiectasia mutated phosphorylation and induction of the NKG2D ligand MIC, were activated to prevent tumorigenesis.     

Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer

Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

Hyperthermia can differentially modulate the repair of doxorubicin-damaged DNA in normal and cancer cells

Hyperthermia can modulate the action of many anticancer drugs, and DNA repair processes are temperature-dependent, but the character of this dependence in cancer and normal cells is largely unknown. This subject seems to be worth studying, because hyperthermia can assist cancer therapy. A 1-h incubation at 37 degrees C of normal human peripheral blood lymphocytes and human myelogenous leukemia cell line K562 with 0.5 microM doxorubicin gave significant level of DNA damage as assessed by the alkaline comet assay. The cells were then incubated in doxorubicin-free repair medium at 37 degrees C or 41 degrees C. The lymphocytes incubated at 37 degrees C needed about 60 min to remove completely the damage to their DNA, whereas at 41 degrees C the time required for complete repair was shortened to 30 min. There was also a difference between the repair kinetics at 37 degrees C and 41 degrees C in cancer cells. Moreover, the kinetics were different in doxorubicin-sensitive and resistant cells. Therefore, hyperthermia may significantly affect the kinetics of DNA repair in drug-treated cells, but the magnitude of the effect may be different in normal and cancer cells. These features may be exploited in cancer chemotherapy to increase the effectiveness of the treatment and reduce unwanted effects of anticancer drugs in normal cells and fight DNA repair-based drug resistance of cancer cells.     

Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (B?cker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.     

DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses

Currently, the potential genotoxicity of high power microwave pulses (HPMP) is not clear. Using the alkaline single cell gel electrophoresis assay, also known as the alkaline comet assay, we studied the effects of HPMP (8.8 GHz, 180 ns pulse width, peak power 65 kW, pulse repetition frequency 50 Hz) on DNA of human whole-blood leukocytes and isolated lymphocytes. The cell suspensions were exposed to HPMP for 40 min in a rectangular waveguide. The average SAR calculated from the temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The steady-state temperature rise in the 50 microl samples exposed to HPMP was 3.5 +/- 0.1 degrees C. In independent experiments, we did not find any statistically significant DNA damage manifested immediately after in vitro HPMP exposure of human blood leukocytes or lymphocytes or after HPMP exposure of leukocytes subsequently incubated at 37 degrees C for 30 min. Our results indicate that HPMP under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites, and incomplete excision repair sites, which could be detected by the alkaline comet assay.     

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.     

Polymorphisms in the XPG gene and risk of gastric cancer in Chinese populations

DNA repair genes play an important role in maintaining stability and integrity of genomic DNA. Polymorphisms in nucleotide excision repair genes may cause variations in DNA repair capacity phenotype and thus contribute to cancer risk. In this case-control study of 1,125 gastric cancer cases and 1,196 cancer-free controls, we investigated the association between three functional single nucleotide polymorphisms (SNPs, rs2296147T > C, rs2094258C > T and rs873601G > A) in the xeroderma pigmentosum group G (XPG) gene and gastric cancer risk. We used the Taqman assays to genotype these three SNPs and logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). We found that only the rs873601A variant genotypes were associated with a significant higher risk for gastric adenocarcinoma (adjusted OR = 1.30, 95% CI = 1.03-1.64 for AA vs. GG and adjusted OR = 1.23, 95% CI = 1.01-1.49 for AA vs. GG/AG). Stratification analysis indicated that this risk was more pronounced in subgroups of older age (>59 years), males, ever-smokers, and patients with NGCA. All these were not found for the other two SNPs (rs2296147T > C and rs2094258C > T). We then performed expression analysis using gastric cancer adjacent normal tissues from 141 patients and found that the A variant allele was associated with non-significantly reduced expression of XPG mRNA (P(trend) = 0.107). Further analysis using mRNA expression data from the HapMap suggested that the A allele was associated with significantly reduced expression of XPG mRNA in normal cell lines for 45 Chinese (P(trend) = 0.003) as well as for 261 subjects with different ethnicities (P(trend) = 0.001). These support the hypothesis that functional XPG variants may contribute to the risk of gastric cancer. Larger studies with different ethnic populations are warranted to validate our findings.