Jana Slyskova

Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER) in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which twelve reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analysed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.

Mismatch repair (MMR) genes are known to be frequently altered in colorectal cancer (CRC). Both genetics and epigenetics modifications seems to be relevant in this phenomenon, however it is still not clear how these two aspects are interconnected. The present study aimed at characterizing of epigenetic and gene expression profiles of MMR genes in sporadic CRC patients from the Czech Republic, a country with one of the highest incidences of this cancer all over Europe.

DNA repair is crucial in maintaining universal genomic stability and preserving cellular functions. Defects in the DNA repair increase vulnerability to endogenous and exogenous DNA-damaging agents, consequent accumulation of mutations in the genome leading to development of several disorders. Individuals with defective DNA repair are at severely increased risk of developing cancer and other diseases. A number of factors are hypothesized to influence individual DNA repair capacity. Measurement of DNA repair capacity, through functional assays, therefore, represents an integrated marker that incorporates the effects of epigenetic factors, genetic polymorphisms, gene expression, stability of gene product, effect of inhibitors/stimulators, environmental factors and lifestyle factors. Functional DNA repair assays reflect the capacity of an organism to cope with a chronic exposure to numerous environmental and dietary genotoxicants and may be used as predictive markers in cancer therapy. We, recently, developed functional assays to measure nucleotide excision (NER) and base excision (BER) repair capacities in human tissues. Our initial data showed that in healthy subjects, occupationally exposed to potential chemical carcinogens, BER capacity inversely correlated with the levels of DNA damage and showed an increase with increased exposure. In contrast, both BER and NER capacities were significantly lower and DNA damage higher in incident colorectal cancer patients than in matched healthy control subjects. In colorectal cancer patients, interestingly, the DNA repair capacity increased to the levels observed in healthy control subjects following the completion of chemotherapy. In our other study, BER and NER capacities were measured in blood cells, healthy mucosa and tumor tissues from 70 patients with sporadic CRC. Lymphocytes in contrast to tumor tissues exhibited highest levels of BER and NER capacities. The definition of genetic and phenotypic landscape of the disease is, thus, further extended by DNA repair functional characteristics, both on the surrogate (systemic) and target tumor (topical level) tissues. Additional studies on DNA repair efficiency to determine influence on onset of the disease and in the context of gene-environment interactions are warranted. Acknowledgement: CZ GA CR: GA P304/10/1286 and GA P304/12/1585 Citation Format: Pavel Vodicka, Jana Slyskova, Rudolf Stetina, Ludmila Vodickova, Alessio Naccarati, Rajiv Kumar, Kari Hemminki. Functional DNA repair tests and their perspectives in molecular epidemiology (experience with subjects exposed to carcinogens and in cancer patients). [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 23.

Purpose: DNA repair capacity (DRC) is a determinant not only of cancer development but also of individual response to therapy. Previously, altered base and nucleotide excision repair (BER and NER) have been described in lymphocytes of patients with sporadic colorectal cancer. We, for the first time, evaluate both excision repair capacities in human colon biopsies to study their participation in colorectal tumorigenesis. Experimental design: Seventy pairs of tumor and adjacent healthy tissues were analyzed for BER- and NER-specific DRC by a comet repair assay. Tissue pairs were further compared for expression levels of a panel of 25 BER and NER genes complemented by their promoter methylation status. Results: We observed a moderate increase of NER-DRC (P = 0.019), but not of BER-DRC in tumors. There was a strong correlation between both tissues for all investigated parameters (P < 0.001). However, 4 NER (CSB, CCNH, XPA, XPD) and 4 BER (NEIL1, APEX1, OGG1, PARP1) genes showed a 1.08- to 1.28-fold change difference in expression in tumors (P < 0.05). Individual gene expression levels did not correlate with overall DRC, and we did not detect any aberrant methylation of the investigated genes. Conclusions: Our complex analysis showed that tumor cells are not deficient in BER and NER, but rather follow patterns characteristic for each individual and are comparable with adjacent tissue. Alteration of excision repair pathways is not a pronounced event in colorectal carcinogenesis. This study shows the feasibility of DRC evaluation in human solid tissues, representing a complex marker of multigene DNA repair processes. Clin Cancer Res; 18(21); 5878–87. ©2012 AACR.

Mutagenesis (2012) 27 (2): 225–232