2387 Immortalization of human oral keratinocytes with Bmi-1 and HPV16-E6 protein
R.H.-K. KIM1, M.K. KANG2, K.-H. SHIN3, Z.M. OO4, T. HAN5, M.A. BALUDA4, and N.-H. PARK2, 1UCLA School of Dentistry, Los Angeles, CA, USA, 2University of California Los Angeles, USA, 3UCLA School of Dentistry CHS43-033, Los Angeles, CA, USA, 4University of California - Los Angeles, USA, 5UCLA, Los Angeles, CA, USA

Objectives: We previously found a strong association between Bmi-1 overexpression and cellular transformation of oral epithelial cells. The current study was undertaken to investigate the cooperative effects of Bmi-1 and human papillomavirus (HPV) viral oncogenes on phenotypic and molecular alterations in normal human oral keratinocytes (NHOK). Methods: Primary NHOK were infected with retroviruses expressing Bmi-1 and selected with puromycin. The drug-resistant cells (HOK/Bmi-1) were superinfected with retroviruses expressing HPV16-E6 or -E7. These cells were maintained in serial subcultures and used for comparison of in vitro replicative capacity, telomerase activity, and the expression levels of cell cycle regulatory proteins, e.g., p53 and pRb. Results: Exogenous Bmi-1 significantly enhanced the replicative capacity of NHOK but did not result in cellular immortalization, which occurred only in combination with expression of E6, but not E7. To identify the E6 functional domain responsible for cellular immortalization, HOK/Bmi-1 cells were superinfected with retroviral vectors expressing the E6 mutants (E6£GSAT, E6£G118-122, and E6£G146-151). Expression of E6£GSAT and E6£G146-151, but not E6£G118-122, allowed for immortalization of HOK/Bmi-1, indicating that the second zinc-finger (ZF) domain corresponding to a.a. 118-122 is critical for the immortalization capability of E6. During the extended life span, telomerase activation did not occur in the HOK/Bmi-1 cells with or without expression of E6 variants until the cells became immortalized. Expression of wild-type E6 or E7 in HOK/Bmi-1 cells resulted in the diminution of protein levels of p53 and pRb, respectively. Interestingly, HOK/Bmi-1 cells immortalized with E6xSAT demonstrated intact p53 protein expression, suggesting that p53 degradation by E6 was not necessary for immortalization. Conclusions: These findings indicate the cooperative effects of Bmi-1 and E6 in cellular immortalization of NHOK, which requires the second ZF domain of E6 and telomerase activation. This study was supported by NIDCR/NIH (DE15316, DE14147, and DE07296) and Oral and Maxillofacial Surgery Foundation.

Seq #240 - Premalignant Oral Lesions and Oral Cancer
3:30 PM-4:45 PM, Friday, March 23, 2007 Ernest N. Morial Convention Center Exhibit Hall I2-J

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