Abrogation of the Retinoblastoma Tumor Suppressor Checkpoint During Keratinocyte Immortalization Is Not Sufficient for Induction of Centrosome-mediated Genomic Instability

ABSTRACT Deregulation of the retinoblastoma (pRB) tumor suppressor pathwayand telomerase activation have been identified as rate-limiting steps forimmortalization of primary human epithelial cells. However, additionalmolecular aberrations including p53 inactivation, ras activation, and de-regulation of protein phosphatase 2A activity are necessary for full trans-formation of immortalized epithelial cells. Genomic instability is observedin most human tumors and constitutes an important mechanism to allowemerging tumor cells to acquire additional mutations to efficiently over-come selection barriers during carcinogenic progression. In an attempt tomodel oral cancer in a human cell-based system, we analyzed normal oralepithelial keratinocytes with the pRB pathway dysregulated by loss ofexpression of the cyclin-dependent kinase (cdk) 4/cdk6 inhibitor p16 INK4A and/or ectopic expression of cdk4 or expression of the human papilloma-virus (HPV) type 16 E7 oncoprotein. Ectopic expression of cdk4 andHPV-16 E7 was equally efficient in extending the life span of normal oralkeratinocytes, and each was able to cooperate with telomerase (hTERT) toimmortalize these cells. HPV-16 E7/hTERT-immortalized normal oralkeratinocytes showed centrosome abnormalities, whereas populations ofcdk4/hTERT-immortalized cells or hTERT-immortalized cells that hadlost expression of p16