Methylation status of tumor related genes during immortalization of human cervical epithelial cell lines

Abstract

Methylation is an epigenetic change in gene function that occurs without a change in the DNA sequence. It is a crucial process during tumourigenesis as multiple tumour suppressor genes are methylated in various cancers. It has been suggested that immortalization is a prerequisite for cancer development, which usually involves a combination of genetic and epigenetic alterations leading to inactivation of tumour suppressors and activation of oncogenes. Tumour suppressor genes (TSGs) are usually silenced by homozygous deletion, transcriptional inactivation or promoter hypermethylation. Studies on deletion and transcriptional inactivation of TSGs during immortalization are well established. However, the role of methylation during immortalization is largely unknown. In this study, the methylation status of a number of tumour-related genes was screened in immortalized cervical epithelial cells using methylation specific PCR (MSP). Preliminary data suggested that the promoter regions of E-Cadherin, RARβ2 (Retinoic Acid Receptor β2), hMSH3 (MutS Homolog 3) and FHIT (Fragile Histidine Triad Gene) were partially methylated in cervical epithelial cell lines immortalized with HPV E6E7. Among these genes, only RARβ2 showed promoter hypermethylation in the early stage of immortalization; it also had the highest frequency of methylation in cervical cancer cell lines. This suggests that RARβ2 may play an important role in cervical carcinogenesis. Three CpG loci MINT1, MINT32 and MINT31 were also examined, and MINT1 and MINT31 showed complete methylation in four cervical carcinoma cell lines but were partially methylated or unmethylated in the immortalized cell lines, suggesting that methylation of these sites maybe a crucial step in cervical carcinogenesis.