Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

Activation of telomerase, a ribonucleoprotein complex that synthesizes telomere repeat sequences, is linked to cell immortalization and is characteristic of most cell lines and tumours. Here we show that expression of the human papillomavirus type 16 (HPV-16) E6 protein activates telomerase in early-passage human keratinocytes and mammary epithelial cells. This activation was observed in cells pre-crisis, that is, before they became immortal, and occurred within one passage of retroviral infection with vectors expressing HPV-16 E6. Studies using HPV-16 E6 mutants showed that there was no correlation between the ability of the mutants to activate telomerase and their ability to target p53 for degradation, suggesting that telomerase activation by HPV-16 E6 is p53 independent. Keratinocytes expressing wild-type HPV-16 E6 have an extended lifespan, but do not become immortal, indicating that telomerase activation and E6-mediate degradation of p53 are insufficient for their immortalization. These results show that telomerase activation is an intrinsic, but insufficient, component of transformation by HPV.

Patients with human papillomavirus associated (HPV+) head and neck cancer (HNC) demonstrate significantly improved survival outcome compared to those with HPV− negative (HPV−) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV− HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV− 0.59 vs. HPV+ 0.22, p<0.0001), corresponding with a prolonged G2/M cell cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%, p=0.002). A genome-wide microarray was used to compare gene-expression 24 hours following radiation between HPV+ and HPV− cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6 fold increase in TP53 (p<0.0001). Using immortalized human tonsillar epithelial cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV− HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.