Radioprotection of oral mucosa by Bmi-1

OBJECTIVES: The long term goal of our research is to develop novel chemopreventive agents for oral mucositis in patients exposed to ionizing radiation (IR). Epithelial tissue damage by radiation is attributed to premature senescence. We have extensively studied the senescence process in normal human oral keratinocytes (NHOK). NHOK senesce in vitro after completion of 22 divisions. Bmi-1, a stem cell factor, greatly enhanced the replicative capacity of NHOK by delaying senescence. We tested whether Bmi-1 could also rescue NHOK from IR-induced premature senescence. METHODS: Exogenous Bmi-1 was transduced in NHOK with retroviral vectors expressing Bmi-1 or the empty vector. The infected cells expressing Bmi-1 (NHOK-Bmi-1) and the control cells (NHOK-B0) were exposed to IR. We compared the phenotypic and molecular alterations between NHOK-B0 and NHOK-Bmi-1 cells after radiation. RESULTS: The vast majority of NHOK-B0 cells underwent premature senescence within 5 days post-IR. These cells stopped proliferation and expressed senescence-associated b-galactosidase (SA b-Gal) activity, which is the marker of senescence. However, NHOK-Bmi-1 cells continued to proliferate, maintained the clonogenic properties, and exhibited reduced staining for SA b-Gal even after 10 Gy. Since cells' ability to cope with genotoxic stress in part depends on DNA repair capacity, we determined the role of Bmi-1 in DNA repair. NHOK-Bmi-1 demonstrated enhanced in vitro DNA end joining activity and in vivo double-strand break repair activity compared to those of NHOK-B0. Since oral mucositis is an inflammatory disorder, we determined the NF-kB-dependent pro-inflammatory signaling in NHOK-B0 and NHOK-Bmi-1 cells. Strikingly, the transcriptional activity of NF-kB was significantly reduced in NHOK-Bmi-1 cells compared to the control. CONCLUSION: These results indicate that Bmi-1 protects oral epithelial cells from IR-induced mucositis by mitigating the genotoxic stress and pro-inflammatory signaling. This study was supported in part by the grants (K22DE15316, R01DE14147, R01DE18295 and K02DE18959) from NIDCR/NIH.