title: Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers.
abstract: 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 12 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 analyzed 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.
authors.affiliation: Department of Molecular Biology of Cancer, Institute of Experimental Medicine ASCR Prague, Czech Republic ; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University Prague, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Slyskova
authors.affiliation: Centre for Brain Ageing and Vitality, Institute for Ageing and Health, Human Nutrition Research Centre, Newcastle University Newcastle upon Tyne, UK ; Environmental Risk and Health Unit, Flemish Institute for Technological Research (VITO) Mol, Belgium.
authors.forename: Sabine A S
authors.initials: SA
authors.lastname: Langie
authors.affiliation: Department of Nutrition, University of Oslo Oslo, Norway.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Collins
authors.affiliation: Department of Molecular Biology of Cancer, Institute of Experimental Medicine ASCR Prague, Czech Republic ; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Vodicka
keywords: base excision repair; human solid tissue; in vitro comet-based DNA repair assay; methodological report; nucleotide excision repair