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. 2001 May;75(9):4467-72.
doi: 10.1128/JVI.75.9.4467-4472.2001.

Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein

T Veldman  1 , I Horikawa, J C Barrett, R Schlegel
Affiliations

Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein

T Veldman et al. J Virol. 2001 May.

Abstract

The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5' promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (-211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

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Figures

FIG. 1
FIG. 1
Telomerase activity in HFKs transduced with HPV-16 E6, E7, or E6 plus E7 genes. Using a modified TRAP assay (17, 35), telomerase activity was detected in HFKs expressing E6 alone or E6 plus E7 but not in HFKs expressing E7 alone. Control HFKs (either nontransduced or transduced with vector LXSN) were also negative for telomerase activity. HeLa (HPV-18-positive cervical cancer line) and IMR-90 (normal embryonic lung fibroblasts) cells were used as telomerase-positive and -negative controls, respectively. HeLa lysates were treated with either RNase A or heat prior to the telomerase reaction to demonstrate telomerase-specific activity.
FIG. 2
FIG. 2
RT-PCR and RNase protection analyses for telomerase hTERT mRNA expression. The transduced keratinocytes described for Fig. 1 were evaluated as follows. (A) RT-PCR analysis was performed using the Superscript preamplification system (Gibco-BRL) and an hTERT-specific primer pair (26). hTERT RNA was observed only in HFKs expressing E6 alone or E6 plus E7. 36B4 primers were used to ensure cDNA integrity and to control for sample loading (21). (B) RNase protection analysis was performed on 40 μg of cellular RNA from the same cells as in panel A using radiolabeled hTERT- and 36B4-specific riboprobes and the RPA II kit (Ambion). Protected fragments are indicated by arrows. Yeast RNA was used to verify probe specificity. The 145-bp hTERT protected fragment was observed only in cells expressing E6 alone or E6 plus E7, confirming the results in panel A.
FIG. 3
FIG. 3
E6 activation of the hTERT promoter and identification of the minimal promoter region necessary for induction. hTERT promoter fragments (solid bars) were cloned into the pGL3-Basic vector (Promega) upstream of the firefly luciferase gene (hatched lines) as described previously (15) and used in luciferase assays. Dotted lines indicate a 35-bp deleted region. Reporter plasmids are named according to the first nucleotide number at the 5′ end of each hTERT promoter fragment. Telomerase-negative keratinocytes were transiently cotransfected with an hTERT luciferase reporter plasmid, an E6 expression vector, or an empty vector and with the pRL-CMV R. reniformis reporter plasmid (to control for transfection efficiency). Relative fold activation (shown on the right) reflects the normalized luciferase activity induced by E6 or empty vector compared to the normalized activity of the largest hTERT reporter plasmid (pGL3B-1125) plus vector control. Error bars show the standard deviations for at least three independent experiments.
FIG. 4
FIG. 4
Immunoblot analyses of cellular Myc and Mad proteins in transduced HFKs. Protein extracts of the transduced keratinocytes used for Fig. 1 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nylon filters, and incubated with either Myc 9E10 monoclonal antibody (Pharmingen; 2 μg/ml) or Mad polyclonal antibody (Pharmingen; 1:1,000 dilution). The specified proteins were detected using alkaline phosphatase-conjugated goat anti-mouse or -rabbit immunoglobulin G antibodies (Tropix; 1:5,000 dilution) and visualized using CDP-Star chemiluminescent substrate (Tropix). To ensure equal loading of protein, immunoblots were stripped and reprobed using an actin monoclonal antibody (Amersham; 5 to 10 μg/ml).
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