The aim of this study was to determine the methylation status of the p16INK4a, p14ARF and p15INK4b genes and the subsequent effect of hypermethylation on the expression of these genes in cervical cancer patients. Methylation-specific PCR (MSP) was performed to analyse the methylation status of p16INK4a, p14ARF and p15INK4b genes and was confirmed by sequencing. Reverse transcription PCR (RT-PCR) was carried out to determine changes in the expression of the genes due to hypermethylation. Hypermethylation of the p16INK4a, p14ARF and p15INK4b gene promoters was observed in 36, 8.8 and 11.2%, respectively, of 125 cervical cancer samples from a north Indian population. Methylation of p16INK4a was significantly (P<0.001) associated with the cervical cancer cases. Significant association of p16INK4a hypermethylation with passive smoking and oral contraceptive use was also observed. Methylation of p15INK4b was also found to be significant (P<0.05). Our findings did not reveal any correlation between the promoter methylation of p16INK4a, p14ARF and p15INK4b with factors, including age and human papillomavirus infection. mRNA expression was significantly reduced in patients with a methylated promoter (P<0.001) of p16INK4a compared to patients with an unmethylated promoter. In conclusion, this is the first study demonstrating significant hypermethylation of p15INK4b and p16INK4a genes among cervical cancer patients in a north Indian population, and a significant association of p16INK4a hypermethylation with passive smoking and oral contraceptive use.

Aberrant methylation of the promoter CpG island of human genes is an alternative gene inactivation mechanisms that contributes to the carcinogenesis of human tumors. We tried to determine the methylation status and its impact on the expression of two tumor related genes Casp-8 and Rb1 in 103 bladder tumor tissues and 48 control paraffin-embedded tissues by using MSP-PCR and SQRT-PCR. Of the patients, 19.4% for Casp-8 and 28.2% for Rb1 showed methylation in bladder cancer. There were significant differences between patients and healthy controls in methylation of Rb1 (p = 0.001) and Casp-8 (p = 0.008) and especially when both genes methylated (p = 0.004). Methylation of Casp-8 has mostly been taken places in patients with age >60 years (p = 0.013) whereas methylation of Rb1 has taken place in age >60 (p = 0.018) as well as in patients age 30 per week (OR = 4.57, 95% CI = 2.38–8.80, p = 0.000). Significant reduction in expression has been detected in patients with methylated Rb1 (p = 0.00) and Casp-8 (p = 0.03). These results suggest that age, smoking and drinking will increase the probability of methylation of these genes and consequently increased risk of developing of bladder cancer to higher stages of disease. Interestingly, it has been deduced that methylation by itself maybe significantly have a role on reducing the expression ofRb1, but it seems that methylation along with risk factors lead to decrease the expression of Casp-8. Methylation of Rb1 can be considered as one of prognosis indicator for progression and development bladder cancer.