hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

<p>Abstract</p> <p>Background</p> <p>Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.</p> <p>Methods</p> <p>Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.</p> <p>Results</p> <p>We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.</p> <p>By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.</p> <p>Conclusions</p> <p>Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.</p

HPV-mediated transformation of the anogenital tract

Infection with high-risk human papillomavirus (HR-HPV) has been associated with intraepithelial neoplasia and carcinomas at various sites of the anogenital tract, including the cervix, vulva, vagina, penis and anus. Although HR-HPV is a necessary cause for cervical cancer, the majority of anal cancers and a subset of cancers at other genital sites, additional (epi)genetic events are required for malignant transformation. HPV-mediated transformation of human epithelial cells has been recognized as a multi-step process resulting from deregulated transcription of the viral oncogenes E6 and E7 in the proliferating cells. Interference of E6 and E7 with cell cycle regulators induces genetic instability, which drives the continuous selection of oncogenic alterations providing cells with a malignant phenotype. Early genetic events during cervical carcinogenesis associated with immortalization, include deletions at chromosomes 3p, 6 and 10p, whereas amongst others gain of chromosome 3q, loss of chromosome 11 and epigenetic alterations such as inactivation of the TSLC1 tumor suppressor gene represent later events associated with tumor invasion