Cervical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levels of oncoprotein expression but exhibit progressive, major increases in hTERT gene expression and telomerase activity

Abstract

Cervical carcinoma cells display high telomerase activity and usually contain and express integrated copies of the human papillomavirus (HPV) genome. Recent studies have demonstrated that the E6 oncogene of malignancy-associated HPVs increases cellular telomerase activity, predominantly via transcriptional activation of the catalytic subunit of telomerase, hTERT. To examine the relationship between E6 oncoprotein expression and telomerase expression during cellular immortalization, we transduced primary human cervical epithelial cells with the HPV E6/E7 genes and monitored temporal changes in viral oncoprotein expression, cellular hTERT RNA expression, and cellular telomerase activity. Quantitation of the individual E6 and E7 proteins, using a newly developed immunoprecipitation/immunoblotting technique, demonstrated that both oncoproteins were expressed at stable levels during successive passages of cervical cells. In contrast, the levels of hTERT mRNA and telomerase activity increased progressively and dramatically during passaging. Late-passage immortalized cells (passage 30) showed a 25-fold increase in hTERT mRNA and a 300-fold increase in telomerase activity compared to early-passage (passage 4) cells. Thus, neither hTERT mRNA expression nor telomerase activity are directly proportional to the level of E6 oncoprotein, indicating that E6 is not the sole determinant of the high levels of telomerase in cervical cells during immortalization.

Figure 1.

Immunoblot analysis of E6 and E7 oncoproteins in transduced cervical epithelial cells. At the indicated passage number, protein extracts were prepared from cervical cells that had been transduced with the HPV-16 E6/E7 oncogenes. The lysates were immunoprecipitated and immunoblotted with the respective antibodies for epitope-tagged E6 (A) and E7 (B) as described in Materials and Methods. E6 and E7 proteins were detected only in cells transduced with the viral oncogenes and appeared stable throughout passaging.

Figure 2.

Telomerase hTERT mRNA expression in E6/E7-transduced cervical cells as determined by qualitative and quantitative RT-PCR. A: Qualitative RT-PCR. Levels of hTERT mRNA were evaluated in the transduced cells via RT-PCR as described in Materials and Methods. Primers were also used to detect control ribosomal 36B4 mRNA to ensure cDNA integrity and to control for sample loading. hTERT RNA expression was observed in high-risk HPV-16 E6/E7-transduced cells but not in low-risk HPV-6 E6/E7 cells or in control LXSN cells. The HPV-16 E6/E7 cells showed a progressive increase in hTERT mRNA during cell passaging (passage 4 to passage 30). B: Quantitative RT-PCR. To verify the above results, temporal changes in the expression of telomerase hTERT mRNA were also determined by real-time quantitative RT-PCR. Levels of hTERT mRNA were evaluated in ectocervical cells transduced with retrovirus LXSN (control), HPV-6 E6/E7, or HPV-16 E6/E7. The primers and methods for this analysis are described in detail in Materials and Methods. Error bars indicate the SD of duplicate PCR reactions. High-risk HPV-16 E6/E7 induced an acute 20-fold increase in hTERT expression (LXSN versus HPV-16 E6/E7 passage 1) that further increased up to 500-fold at the stage of immortalization (LXSN versus HPV-16 E6/E7 passage 20).

Figure 3.

Telomerase activity in E6/E7-transduced cells. To verify that the increased hTERT expression observed in Figure 2 ▶ resulted in increased telomerase activity, the same cells were extracted and 5 μg of total protein was assayed by TRAP assay. Telomerase activity was detected in HPV-16 E6/E7 cells but not in control LXSN cells or cells transduced with HPV-6 E6/E7. HeLa cells (HPV-18-positive cervical cancer cell line) and IMR-90 cells (normal embryonic lung fibroblasts) served as telomerase-positive and telomerase-negative controls, respectively. Serial dilutions of lysates from HPV-16 E6/E7-expressing cells were assayed to provide semiquantitative estimates of telomerase activity (A, 5 μg; B, 1.5 μg; C, 0.15 μg; and D, 0.015 μg). HPV-16 E6/E7 cells showed a 300-fold increase in telomerase activity between passages 4 and 30 (compare similar activities of lane 4 of A with lane 7 of D).