hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis.
BACKGROUND
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.


METHODS
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.


RESULTS
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.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.


CONCLUSIONS
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.
de Wilde
Jillian
J
Department of Pathology, Unit of Molecular Pathology, VU University Medical Center, PO box 7057, 1007 MB Amsterdam, the Netherlands.
Kooter
Jan M
JM
Overmeer
Renée M
RM
Claassen-Kramer
Debbie
D
Meijer
Chris J L M
CJ
Snijders
Peter J F
PJ
Steenbergen
Renske D M
RD
D024509:3' Flanking Region; D024506:5' Flanking Region; D002472:Cell Transformation, Viral / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D005091:Exons; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D052162:Human papillomavirus 16 / Q000235:genetics; D052161:Human papillomavirus 18 / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology* / Q000821:virology; D030361:Papillomavirus Infections / Q000150:complications* / Q000821:virology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology / Q000821:virology
D012333:RNA, Messenger; C509186:TERT protein, human; D019098:Telomerase