Methylation of Cervical Neoplastic Cells Infected With Human Papillomavirus 16

Objective This study was conducted to evaluate the role of methylation of adenylate cyclase activating peptide 1 (ADCYAP1), paired box gene 1 (PAX1), cell adhesion molecule 1 (CADM1), and T-lymphocyte maturation-associated protein (MAL) during carcinogenesis. Methods We evaluated the methylation of 4 genes by using the cervical carcinoma cell lines (CaSki, SiHa, HeLa, and C33A) and cervical neoplastic cells from 56 subjects with human papillomavirus 16 (HPV16)-infected low-grade squamous intraepithelial lesions (LSILs), 50 subjects with HPV16-infected high-grade squamous intraepithelial lesions (HSILs), and 24 subjects with HPV16-infected invasive cervical cancer who attended Seoul St. Mary's Hospital. Methylation of the 4 genes was evaluated using quantitative bisulfate pyrosequencing. Results The ADCYAP1 promoter was hypermethylated in the 4 cell lines (CaSki, 97.40 1.39; SiHa, 82.04 +/- 17.02; HeLa, 96.14 +/- 2.08; and C33A, 78 +/- 10.18). PAX1 and CADM1 were hypermethylated in the HPV16/18-infected cell lines CaSki (PAX1, 91.18 +/- 9.91; CADM1, 93.5 +/- 7.33), SiHa (PAX1, 96.14 +/- 2.08; CADM1, 93.15 +/- 8.81), and HeLa (PAX1, 82.04 +/- 17.02; CADM1, 92.43 +/- 9.95). MAL was hypermethylated in the CaSki cell line (96.04 +/- 4.74). Among human cervical neoplastic cells, the methylation indices of ADCYAP1 were 7.8 (95% confidence interval [95% CI], 7.0-8.6) in subjects with LSILs and 39.8 (95% CI, 29.0-54.7) in those with cervical cancer (P < 0.001); for PAX1, 7.2 (95% CI, 6.1-8.5) and 37.8 (95% CI, 27.1-52.7), respectively; for CADM1, 3.5 (95% CI, 3.0-4.0) and 17.7 (95% CI, 10.8-29.1), respectively; for MAL, 2.7 (95% CI, 2.5-3.0) and 13.0 (95% CI, 7.6-22.0), respectively (P < 0.001 for each). Immunohistochemical staining results were positive in the cytoplasm of subjects with low methylation of the 4 gene promoters; however, they were negative in the cytoplasm of those with hypermethylation of the 4 gene promoters. Conclusions The results of this study suggest that the methylation of ADCYAP1, PAX1, CADM1, and MAL may be highly associated with the development of cervical cancer, and that gene expression can be suppressed by gene promoter hypermethylation.