HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium.

The E6 and E7 genes of HPV-16 or HPV-18 both are necessary for effective immortalization of primary human genital keratinocytes. To analyse the individual role of E6 and E7 genes in dysregulating cell growth, we cloned the HPV-16 E6, E7 and E6/E7 genes into retroviruses. Primary human keratinocytes (PHK) were then infected with these retroviruses and selected in differentiation-inducing medium (high calcium and serum). The E6/E7 retroviruses were the most effective at inducing differentiation-resistant colonies. Intermediate numbers of colonies were induced by E6 and low numbers by E7. Interestingly, only cultures infected with E7 and E6/E7 retroviruses showed a significant proportion of cells progressing into the S phase, consistent with our earlier studies showing that E7 is required for the efficient immortalization of genital keratinocytes. Accompanying this entry into S phase, the E7 or E6/E7 transduced cells expressed high levels of cyclins A, B and E, but lower levels of cyclin D. In addition, cdc-2, cdk-2 and cdk-4 were also increased. No significant differences were detected in the expression of c-myc and c-fos between the vector and any of the transduced cells. Keratinocytes infected with the E7 retrovirus exhibited decreased levels of Rb protein and increased levels of p53, whereas cells infected with E6-expressing retroviruses displayed normal levels of Rb protein and decreased levels of p53. Finally, E7 induced a three-fold increase in bcl-2 expression. Our results indicate that the HPV-16 E7 gene alone is sufficient to bypass keratinoctye growth arrest induced by serum and calcium exposure and that the discordant expression of several cell regulatory proteins accompanies this unregulated proliferation.