Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation

Abstract

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer

“…Most studies of DNA repair and cancer have been performed in PBLs (15)(16)(17) or in established cell lines derived from metastatic disease (10,21,22), especially the HeLa cell line, which exhibits a level of NER at the upper end of the considerably variable range of human cells (32). In vitro cancer studies rarely contain tissuematched controls as a result of the inability to grow such cells in traditional culture systems.…”

Differences in Repair of DNA Cross-links between Lymphocytes and Epithelial Tumor Cells from Colon Cancer Patients Measured In vitro with the Comet Assay

“…Recently, this assay was modified for measuring nucleotide excision repair (NER) activity (14,15). The repair enzymes present in cell extracts recognize the damage in the DNA of the substrate cells and incise the DNA.…”

“…The strand breaks produced are detected by the conventional comet assay, and the increase in tail DNA (TDNA) reflects the repair activity of the cell extract. Up to now, the studies done have tested lymphocyte extract activity to repair oxidative damage (13), DNA adducts (14), or cyclobutane pyrimidine dimmers (15), but it is unclear if in vitro repair activity in lymphocyte extracts reflects repair capacity in other tissues of the individual (7). Additionally, whether the extract could repair other kinds of damage, such as DNA cross-links induced by cancer drugs, is not assessed.…”

DNA Repair Measured by the Comet Assay

“…In this in vitro approach a substrate, in the form of agaroseembedded nucleoids derived by lysis of cells containing a specific lesion, is incubated with the extract whose excision repair activity is to be measured by the comet assay Gaivão et al, 2009;Langie et al, 2006) (Figure 4). The nature of the DNA lesion in the substrate defines the repair pathway that is measured.…”

“…The nature of the DNA lesion in the substrate defines the repair pathway that is measured. Substrate containing 8-oxoG is used to measure the BER activity of 8-oxoG DNA glycosylase (OGG) in the extracts tested ; if substrate contains bulky adducts or cyclobutane pyrimidine dimers NER is measured (Gaivão et al, 2009;Langie et al, 2006). More recently an assay for crosslink repair has been developed (Herrera et al, 2009).…”