Cigarette smoke-induced DNA damage detected by Comet assay in HPV-transformed cervical cells Free

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Carcinoma of the cervix is one of the leading cancer types among women worldwide. HPV (Human Papilloma Virus) infection is the necessary causative factor in the development and progression of cervical neoplasia in more than 97% of the cases. However, it has been shown that only a small percentage of the HPV-positive women would develop cervical abnormalities. This indicates that HPV infection alone is insufficient, although necessary, in development and progression of cervical neoplasia and cancer. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers. We hypothesize that cigarette smoke-mediated DNA damage plays a significant role in cervical cancer in smokers. Since DNA repair machinery is one of the primary defense mechanisms against mutagen-induced insult to the genome, we assessed the DNA damage and repair capacity in the HPV-16 transformed human ectocervical cell line (ECT E6/ E7). The cells were treated with 20, 40, and 60 μg/ml cigarette smoke condensate (CSC) for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, and 72 h. Concentrations of CSC lower than 20 μg/ml did not induce any DNA damage, while higher than 60 μg/ml had a lethal effect on the cells. The single- and double-strand breaks were measured by alkaline and neutralizing Single Cell Gel Electrophoresis (SCGE) assays, respectively. A total of 50 cells per slide and 2 slides per sample were analyzed by the video image analysis software, Comet Assay IV. Statistical analysis of the data was carried out using one-way ANOVA. The mean tail length was increased by 1.3, 2.4, 3.6 fold (p<0.0001) after treatment with 20, 40 and 60 μg/ml CSC in alkaline assay; the neutralizing assay also revealed a 1.3, 1.7, and 2.6 fold increase (p<0.0001) after subsequent CSC treatment with the above concentrations. Likewise, the tail moment increased dose-dependently (p<0.0001 for both assays). Both single- and double-strand DNA breaks were removed time-dependently during the repair phase of the reaction. However, a substantial amount (51% and 26%) of the single- and double-strand DNA breaks were not removed even after 72 h, respectively. Together, our data reveal high degree of persistence of the DNA damage associated with cigarette smoke constituents in the HPV-16 transformed human ectocervical cells, and suggest that persistence of the cigarette smoke-associated DNA damage in the HPV-infected cervical cells may lead to increased mutation and ultimately higher cancer risk.