Radioprotective Effects of Bmi-1 for Normal Human Keratinocytes

OBJECTIVES: Bmi-1 is a polycomb group protein necessary for the maintenance of stem cell phenotype. The current study was undertaken to determine the role of Bmi-1 in protecting human oral and cutaneous epithelial cells from ionizing radiation (IR). METHODS: Primary normal human keratinocytes (NHKs) were infected with retroviral vectors expressing Bmi-1(RV-Bmi-1) or empty vector (EV). The infected cells were exposed to 0, 5, or 10 Gy IR and assayed for phenotypic alterations, i.e., clonogenic efficiency, cell morphology, and expression of senescence-associated beta-galactosidase (SA b-Gal) activity. RESULTS: NHK infected with EV (NHK/EV) rapidly underwent premature senescence upon exposure to 5 or 10 Gy IR. However, the cells overexpressing Bmi-1 (NHK/Bmi-1) continued to replicate post-IR and demonstrated significantly higher clonogenic efficiency than did the NHK/EV cells. To determine whether the radioresistance of the NHK/Bmi-1 cells was associated with enhanced DNA repair, we compared the DNA repair activities for double stranded breaks (DSBs) in cells with or without Bmi-1 overexpression. In vitro DNA end joining (EJ) activity was notably induced in NHK/Bmi-1 compared with the NHK/EV cells, especially for blunt-ended DSBs. Chromosomal DSB repair activity was also compared between cells expressing Bmi-1 and the controls by pulse-field gel electrophoresis after exposure to 60 Gy IR. Bmi-1 expression in cells led to significant increase in the kinetics of DSB repair in vivo for the IR-induced chromosomal DSBs. Furthermore, exogenous Bmi-1 in primary NHK cultures inhibited the TGF-b signaling pathway, which is strongly activated in cells after exposure to IR and is known to trigger the IR-induced cell growth arrest. CONCLUSIONS: These data indicate that Bmi-1 elicits radioprotective effects in primary NHKs and suggest possible therapeutic use of this protein for protection of oral and cutaneous tissues from radiation damage. This study was supported by the grants from NIDCR/NIH (R01DE18295, R01DE14147, and K22DE15316).