Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action

Abstract

In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

Retinoid‐related molecule AGN193198 potently induces G2M arrest and apoptosis in bladder cancer cells

“…41,42 In particular, exposure of various carcinoma cell lines to ionizing radiation, mutagens and the DNA-damaging agent etoposide results in an accumulation of cells in the G 2 M phase of cell cycle that precedes induction of apoptosis. [43][44][45][46] The chalcone-based structure of AGN193198 47 is somewhat rigid and planar. 39 These properties might allow intercalation and/or binding of AGN193198 to DNA resulting in immediate damage, and, as observed, immediate G 2 M and S phase accumulations that precede the appearance of large numbers of apoptotic cells.…”

Preclinical Evaluation of the Pharmacodynamic Properties of 2,5-Diaziridinyl-3-Hydroxymethyl-6-Methyl-1,4-Benzoquinone

“…The repair of drug induced DNA cross-links is important and can be followed using the Comet assay (51,56). In this study, estimations of DNA cross-linking after 2 hours of treatment followed by a 24 drug-free recovery phase suggests that RH1 adducts are indeed repaired.…”

Dynamic Analysis of DNA Damage by Flow Cytometry and FISH

“…Such information requires the introduction of dynamic cytogenetic and molecular techniques. In the last few years, techniques such as FC, FISH, and single nucleotide polymorphism (SNP) were introduced in the study of the genetic instability induced by noxious agents [18,20,22,27,28,29,30,31]. The introduction of FC in the above studies made the analysis easier and the statistical evaluation more reliable, while the introduction of FISH using centromeric DNA probes [33,34] provided some information on the chromosomal constituency of the induced micronuclei.…”

“…To evaluate their potential for inducing chromosome breaks (clastogenic action) or poisoning mitotic spindle apparatus (aneugenic action), the micronucleus (MN) assay was introduced for both in vivo and in vitro studies [24,25,26]. During the last few years, molecular techniques were introduced in combination to the micronucleus assay for the molecular analysis of genotoxicity [27,28,29,30,31].…”

“…It can be applied to all cell types containing micronuclei [35,36]. Centromeric and telomeric probes were used in FISH studies in an attempt to elucidate the aneugenic or clastogenic way of action of noxious agents on DNA [27,28,29,30,31,37]. Although such probes give us the ability to distinguish between aneugenic and clastogenic action, they do not supply information on the molecular events preceding these actions.…”