Degradation of p53, not telomerase activation, by E6 is required for bypass of crisis and immortalization by human papillomavirus type 16 E6/E7

Abstract

Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.

FIG. 1.

p53 degradation by E6 mutants is necessary and sufficient for immortalization with E7. Primary HFK were infected with retrovirus expressing the indicated E6 mutants, in tandem with E7, at the second absolute passage. Empty vector (pBabe) and wild-type E6/E7 cells were included as controls. Cells were counted after trypsinization and seeded at defined density for each passage. (A) Cumulative PDs are graphed as a function of time in culture, shown as passage number. For cell lines that did not immortalize, icons matching the legend were used to indicate the point at which these cells ceased to grow. The graph represents the averages of three independent experiments. (B) RT-PCR was performed to check for similar E6 and E7 expression in the mutant cell lines. As previously described (18, 93), multiple splice variants of E6 can be detected. Data shown are from line A. Similar results were obtained with lines B and C.

FIG. 2.

Telomerase activity is induced as expected in E6 mutant/E7 keratinocytes over time. TRAP assays were performed to measure telomerase activity for each mutant, over time, indicated as passage number. Immediately following selection, the behavior of each mutant was as expected (Table 1), although levels varied somewhat over the lifetime of each culture. Results from lines A and B are shown. Line C was similar to line A.

FIG. 3.

p53 accumulation and activation accompany cell death at crisis in nonimmortalizing E6 mutant/E7 lines. Western blot assays were performed with 50 μg of total protein from the indicated cell lines, at the indicated passage numbers. Blots were probed for total p21^CIP1 or phospho-p53, modified on serine-15, and then stripped and reprobed for total p53 and p16^INK4a, followed by actin, as a loading control. Data shown are from line B. Similar results were obtained with lines A and C.

FIG. 4.

Telomere lengths shorten over the life span of wild-type and mutant E6/E7 cells. Southern blot analysis was performed for telomere restriction fragment (TRF) length on the pBabe vector control, mutant E6/E7, and wild-type E6/E7 keratinocyte lines. Passage number is indicated at the top of each lane. Average TRF length, indicated at the bottom of each lane, was determined using standard calculation methods (1), employing the formula [Σ(length) · (intensity)]/Σ(intensity) for each lane.