HTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

Jillian De Wilde,Jan M Kooter,Renske Dm Steenbergen,Debbie Claassen-Kramer,Renée M Overmeer,Peter Jf Snijders,Chris Jlm Meijer

doi:10.1186/1471-2407-10-271

Abstract

BackgroundActivation 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.MethodsUsing 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.ResultsWe 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.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.ConclusionsMethylation 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.

Highlights

Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus-induced cervical carcinogenesis
Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in human papillomavirus (HPV)-immortalized cells and cervical cancer cells
Most normal cervical tissues and low-grade cervical intraepithelial neoplasias (CINs) lesions were devoid of detectable hTERT mRNA and telomerase activity [21]. These findings indicate that elevated levels of hTERT mRNA reflect a late step in the CIN to carcinoma sequence following high-risk human papillomavirus (hrHPV) infection

Summary

Introduction

Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. Zinn et al [17] showed that in most cancer cell lines the hTERT promoter is densely methylated, but that CpGs around the transcription start site of a substantial number of hTERT alleles is unmethylated. Choi et al [11] observed in colorectal cancer hypermethylation of a specific CpG outside known transcription factor binding sites, that together with hypermethylation of a number of CpGs closer to the transcription start site was associated with increased hTERT expression It is unknown how methylation of these CpGs induce hTERT expression. The CpG rich region encompassing the 5' proximal region of the gene has been shown to affect hTERT expression [18] This region binds the transcriptional repressor CTCF and in cancer cells CTCF repression was shown to be relieved by methylation of its binding site. Aberrant methylation of the 5' end of gene, including CTCF sites, can affect hTERT expression

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