The development of cancer is a multi-step process that occurs via accumulation of a series of discrete, irreversible, and complementary events. Among these events, the escape of cells from senescence is a critical step, since it could lead to immortalization and tumorigenic transformation of cells. To understand the in-depth mechanisms of cellular senescence and immortalization we investigated the role of Bmi-1, a polycomb group protein identified as c-myc cooperating oncogene in murine lymphomagenesis, in oral carcinogenesis and on the phenotypic changes of normal human oral keratinocytes (NHOK).

First, we determined the status of in situ Bmi-1 expression in human squamous cell carcinomas and human oral preneoplastic tissues using immunohistochemical staining. The level of Bmi-1 was almost negligible in normal tissues, but it was notably increased in preneoplastic oral lesions and cancer tissue, indicating that enhanced Bmi-1 expression is likely associated with oral carcinogenesis and the phenotypic changes of NHOK. Second, we found that an ectopic expression of Bmi-1 extended the lifespan of NHOK in vitro. Extension of life span by Bmi-1 was not associated with enhanced expression of hTERT (human telomerase reverse transcriptase) or repression of p53 and p16^INK4A. We found that Bmi-1 interfered with the TGF-β-mediated growth arrest response in NHOK by inhibiting phosphorylation of Smad2 and led to reduced expression of TGF-β-specific cyclin-dependent kinase inhibitors such asp15^INK4B and p57^KIP2. However, Bmi-1 alone did not result in cellular immortalization of NHOK, which required additional factors, such as human papillomavirus type 16 (HPV-16) E6. HPV-16 E6 expression in NHOK overexpressing Bmi-1 led to immortalization by allowing enhanced expression of hTERT and activation of telomerase. hTERT activation during immortalization occurred by physical interaction of hsp90 protein onto hTERT promoter region, which was necessary for the enhanced hTERT expression in cells.

Together, our findings suggest that NHOK might convert to immortal cells when the cells lose several defensive mechanisms including TGF-β-mediated growth arrest and the loss of telomerase activity, but not p 16^INK4A , p53, or p21^WAF1.