Myc and Human Papillomavirus Type 16 E7 Genes Cooperate To Immortalize Human Keratinocytes

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Abstract
The E6 protein of the oncogenic human papillomaviruses (HPVs), in combination with the E7 protein, is essential for the efficient immortalization of human foreskin keratinocytes (HFKs). Since we recently demonstrated that E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a Myc-dependent mechanism, we speculated that overexpressed Myc might be able to substitute for E6 in cell immortalization. Myc (similar to E6) was unable to immortalize HFKs when transduced alone, despite inducing high levels of telomerase activity. However, when transduced with E7, Myc immortalized HFKs following a brief but detectable crisis period. In contrast to E6 + E7-immortalized cells, the Myc + E7-immortalized cells expressed high levels of p53 protein as well as two p53-regulated proteins, p21 and hdm-2. The increase in p21 and hdm-2 proteins correlated directly with their mRNA levels, suggesting transcriptional activation of the respective genes by the overexpressed p53 protein. Interestingly, a significant proportion of the p53 protein in the Myc + E7-immortalized cells was localized to the cytoplasm, potentially due to interactions with the overexpressed hdm-2 protein. Regardless, cell immortalization by the Myc + E7 genes occurred independently of p53 degradation. Since we have already observed high-efficiency cell immortalization with the hTERT + E7 or E6 mutant (p53 degradation-defective) + E7 genes (i.e., no crisis period) that proceeds in the presence of high levels of p53, we hypothesize that the crisis period in the Myc + E7 cells is due not to the levels of the p53 protein but rather to unique properties of the Myc protein. The common factor in cell immortalization by the three gene sets (E6 + E7, Myc + E7, and hTERT + E7 genes) is the induction of telomerase activity.