Carcinogenesis Advance Access originally published online on September 26, 2008
Carcinogenesis 2008 29(12):2425-2431; doi:10.1093/carcin/bgn225
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Association of hsp90 to the hTERT promoter is necessary for hTERT expression in human oral cancer cells
1 University of California, Los Angeles School of Dentistry, Center for the Health Sciences, 10833 Le Conte Avenue
2 University of California, Los Angeles Dental Research Institute
3 University of California, Los Angeles Jonsson Comprehensive Cancer Center
4 David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA
* To whom correspondence should be addressed. Tel: +1 310 825 8048; Fax: +1 310 794 4001; Email: mkang{at}dentistry.ucla.edu
Enhanced expression of human telomerase reverse transcriptase (hTERT) occurs frequently during cellular immortalization. The current study was undertaken to determine the mechanism regulating the hTERT promoter activity during cellular immortalization of human oral keratinocytes. Normal human oral keratinocytes (NHOKs) were immortalized with Bmi-1 and the E6 oncoprotein of human papillomavirus type 16 to establish the telomerase-positive HOK-Bmi-1/E6 cell line. Using DNA–protein-binding assay, we found that heat shock protein 90 (hsp90) physically interacts with the hTERT promoter in vitro. The hsp90 interaction with the promoter was detected more strongly in the telomerase-positive HOK-Bmi-1/E6 cells compared with that in senescing NHOK. Chromatin immunoprecipitation confirmed the in vivo interaction between hsp90 and the hTERT promoter in SCC4 cells, a telomerase-positive oral cancer cell line, but not in the NHOK. To determine the physiological significance of this interaction, SCC4 cells were exposed to geldanamycin (GA), a competitive inhibitor of hsp90. GA exposure led to decrease in telomerase activity, hTERT promoter activity and hTERT messenger RNA expression in SCC4 cells, even in the absence of de novo protein synthesis. Also, it abolished the in vivo interaction of the hTERT promoter region with hsp90 but not with Sp1 or c-Myc. These results indicate that physical interaction between hsp90 and the hTERT promoter occurs in telomerase-positive cells but not in normal human cells and is necessary for the enhanced hTERT expression and telomerase activity in cancer cells.
Abbreviations: ChIP, chromatin immunoprecipitation; GA, geldanamycin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hsp90, heat shock protein 90; hTERT, human telomerase reverse transcriptase; mRNA, messenger RNA; MS, mass spectrometry; NHOK, normal human oral keratinocyte; OSCC, oral squamous cell carcinoma; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PD, population doubling; PMP, promoter magnetic precipitation; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFIID, transcription factor II D
Received June 9, 2008; revised August 27, 2008; accepted September 18, 2008.