Bmi-1 cooperates with human papillomavirus type 16 E6 to immortalize normal human oral keratinocytes.

Bmi-1 is a member of the polycomb group (PcG) transcription repressors and is implicated in human carcinogenesis. In normal human oral keratinocytes (NHOK), we found that exogenous Bmi-1 expression significantly extended the replicative life span without causing cellular immortalization. Immortalization of NHOK occurs only in combination with human papillomavirus type 16 E6 (HPV-16 E6) but not with E7. During immortalization of NHOK by sequential expression of exogenous Bmi-1 and E6, telomerase activation was observed only after the cells had overcome crisis. Genetic analysis with E6 deletion mutants revealed that the intact second zinc finger domain (amino acids 118-122) was necessary for its cooperative effects with Bmi-1 in the immortalization process. Using these mutants, we found that the increased telomerase activity was closely associated with cell immortalization by Bmi-1 and E6, whereas p53 degradation was not. Using microarray analysis, we identified genes that are immortalization-specific and may participate in the process of NHOK immortalization by Bmi-1 and HPV-16 E6. Our results provide new information on the roles of Bmi-1 and HPV-16 E6 in the multi-step process of oral epithelial carcinogenesis.