Abstract 1700: The nitration of DNA polymerase gamma (pol-gamma) that activates autophagy responses is a novel mechanism by which UVB induces skin cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA polymerase γ (polγ), a major mitochondrial DNA repair enzyme, is essential for replication and repair of the mitochondrial genome. It is well-established that mitochondrial genomic stability is important for cell survival; however, the role of polγ in autophagy remains mysterious. A previous study by our laboratory demonstrated that polγ is inactivated upon UVB radiation by a peroxynitrite-mediated mechanism. In this study, we identified the amino acid residues in the active site of polγ that are targets of nitration-induced inactivation by LC/MS/MS analysis coupled to immunoprecipitation, immuno-blotting and enzymatic activity assays. The results demonstrated that treatment of purified polγ with peroxynitrite leads to the nitration of tyrosine residues in the catalytic domain of polγ resulting the loss of its 3\` to 5\` exonuclease activity. Herein, we also confirmed the presence of nitrated polγ following UVB radiation in mouse skin epithelial cells (JB6) by immunoprecipitation and Western blotting. These results validate the presence of nitrated polγ and the reduction of its enzymatic activity upon UVB treatment in mouse skin. To elucidate the role of polγ in autophagy, JB6 cells were transfected with GFP-labeled LC-3 cDNA and treated with UVB or the wild-type C57BL/6 mice were exposed to 5 kJ/m2 of UVB radiation. The results demonstrated that treatment with UVB increases the LC-3 cleavage and beclin levels suggesting increased autophagic response both in vivo and in vitro. To validate that the increase in autophagic response is due, at least in part, to the inactivation of polγ, polγ in JB6 cells were inactivated by silencing by siRNA for polγ. Consistent with the role of polγ in preventing UVB-induced autophagy, knock down of polγ led to an increase in m-TOR phosphorylation, AKT-phosphorylation and LC-3 cleavage. Treatment of cells with rapamycin, a known inhibitor of m-TOR phosphorylation, blocked the formation of LC-3 cleavage after UVB radiation suggesting that m-TOR activation is associated with UVB-induced autophagy. Our data also showed that the treatment of polγ deficient cells with rapamycin attenuated the polγ siRNA-mediated m-TOR activation and autophagic response suggesting the polγ-mediated autophagy is dependent on m-TOR activation. To further elucidate whether the inactivation of polγ is linked to the progression of skin cancer, we examined the consequences of UVB radiation on cell transformation and invasion in polγ deficient cells. The results show that suppression of polγ alone increases the invasiveness of JB6 cells and treatment with UVB further increases the invasiveness of polγ deficient cells. These results identify tyrosine residues in the active site of polγ as critical targets for its nitrative inactivation and suggest that defective polγ function activates autophagic response to promote UVB mediated carcinogenesis. Citation Format: Sanjit K. Dhar, Vasudevan Bakthavatchalu, Lu Miao, Jing Chen, Haining Zhu, Daret K. St. Clair. The nitration of DNA polymerase gamma (pol-gamma) that activates autophagy responses is a novel mechanism by which UVB induces skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1700. doi:10.1158/1538-7445.AM2013-1700