The comet assay: applications to study cancer risk influenced by external exposure

Bilyk O.O.1, Brieieva O.1, Buchatskiy L.P.2

O. Bilyk, O. Brieieva, L. Buchynska

R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine

IntroductionThe comet assay is a reliable method for measuring DNA damage and repair in human cells such as lymphocytes, epithelial cells, and tumor cells of different origin and has applications in genotoxicity studies, bio-monitoring and cancer research. The potential use of assay as a tool for estimation an individual cancer risk in patients with high predisposition to cancer and clinical management of cancer is widely discussed. Aim: To study DNA damage and repair capacity in epithelial cells and peripheral blood lymphocytes (PBL) after exposure to genotoxic agents in healthy and cancer patients with family history of cancer. MethodsPBL from 45 endometrial cancer (EC) patients were exposed to bleomycin for 30 min. DNA repair capacity was assessed after 15 min incubation in RPMI 1640 without bleomycin. Primary and immortalized by HPV16 E6/E7 oncogenes of ovarian surface epithelial (OSE) cell cultures obtained from 5 women at high risk (HR) of ovarian cancer were exposed to mitomycin C (MMC) at concentration 50 nmol/L. PBL from 10 healthy volunteers and 5 primary OSE cell cultures from women without family history of cancer were evaluated for DNA damage as a control. DNA damage was assessed by comet assay as a mean % of DNA in the tail and the comet tail moment (TM). Results: The level of basal DNA damage in PBL of EC patients was significantly higher (TM 2.1±0.2) compared to healthy volunteers (TM 0.2±0.1). Among EC patients the sensitivity of PBL to genotoxic exposure to bleomycin was higher in patients with family history of cancer (TM 104.9±1.2) then in patients with sporadic cancer (TM 97.3±0.6). It has been found that efficiency of DNA repair in PBL of EC patients depended on family history of cancer. After 15 min repair PBL restored the damaged DNA to the level of TM 55.0±2.4 in EC patients with family history of cancer and up to 22.7±1.1 in patients with sporadic EC. Basal damage of DNA was significantly higher for the primary HR OSE cells than in control OSE cells (TM 15.1±0.1 and 1.0±0.2, respectively). Cell incubation with MMC resulted in progressive increasing of the level of DNA damage of HR OSE cells (TM 25.3±0.2) whereas normal OSE cell line was resistant to genetoxic exposure (TM 1.1±0.5). After immortalization of OSE cells by HPV E6/E7 oncogenes the intensity of DNA damage significantly increased independently of family history of cancer. HR OSE cells immortalized with HPV E6/E7 oncogenes demonstrated increased sensitivity to genotoxic exposure compared to normal immortalized OSE cells (TM 88.5±0.1 and 16.0±0.1, respectively). Conclusion: Enhanced DNA damage of PBL and OSE cells in healthy and cancer patients with family history of cancer under genotoxic and viral oncogenes exposure could expand our understanding of endometrial and ovarian cancer etiology and biology. Comet assay is a useful tool for assessment of individual sensitivity to genotoxic and viral exposure to evaluate the risk of malignancy in patients with predisposition to cancer.

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