Deregulated hTERT transcription during HPV-induced carcinogenesis is dependent on transcriptional and epigenetic alterations

2231 Activation of telomerase resulting from deregulated hTERT expression is a key event during cervical carcinogenesis, a multistep process initiated by an infection with high-risk human papillomavirus (hrHPV) types. In the present study we examined the mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.
 Using luciferase (luc) reporter assays the activity of various hTERT promoter and exonic sequences was analyzed in telomerase negative cells and telomerase positive cells. The latter included HPV16 and HPV18-immortalized keratinocytes, which represent an intermediate state in the carcinogenic process, and cervical cancer cells. In telomerase positive cells, hTERT mRNA expression levels correlated with hTERT promoter-luc activity. However, the hTERT promoter-luc activity was also evident in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In all cells, basal hTERT promoter-luc activity was found to be highly dependent on Sp1 and E-box binding sites.
 Additionally, we showed that transcriptional repression by sequences both upstream and downstream of the core promoter was associated with DNA methylation, as determined with extensive bisulfite sequencing analysis. Methylation at -400 to -200 and +300 to +400 relative to the ATG start site was found to be specifically increased in hTERT positive cells as compared to hTERT negative cells. This indicates that these epigenetic alterations contribute to transcriptional regulation of hTERT in hrHPV-transformed cells. Subsequent analysis of cervical tissue specimens by methylation specific PCR (MSP) targeting both regions showed methylation in virtually all cervical carcinomas, a subset of high-grade precursor lesions, compared with a minority of low grade precursor lesions and normal controls.
 Taken together, we show that both transcriptional regulators like Sp1 and Myc as well as DNA methylation at regions surrounding the core promoter are involved in the regulation of hTERT expression in HPV-infected cells. In addition, methylation of the hTERT promoter and first exon may provide valuable biomarkers for the detection of cervical cancer and its high grade precursor lesions.