Abstract
Abstract
[Purpose] To identify the mechanisms that control the DNA metabolic response to ionic and nonionic radiation in human cells, we investigated this issue using the experimental methods that we originally developed. Recently, we noted the indication that chaperones, such as Heat shock protein 27 (HSP27) and the 78-kDa glucose-regulated protein (GRP78), are important factors involved in these control mechanisms (Exp. Cell Res., 298, 584-592, 2004, Exp. Cell Res., 305, 244-252, 2005). In the present study, we searched for HSP27-interacted proteins to clarify the roles of HSP27 in cellular UVC response. [Methods] The HSP27 protein purified as a fusion protein with GST (GST-HSP27) was conjugated with NHS-activated Sepharose. Cell lysates from the human UVC-resistant cells were applied to the affinity column, and subjected to SDS-PAGE and mass spectrometrical analyses. Interaction of candidate proteins with HSP27 was examined by immunoprecipitation analysis, and the function of the candidate in UVC resistance was further examined using small-interfering RNA (siRNA). [Results and Conclusion] Annexin II was specifically bound to GST-HSP27-conjugated Sepharose compared with GST-conjugated Sepharose. The interaction between HSP27 and annexin II was confirmed by coimmunoprecipitation using anti-HSP27 and anti-annexin II antibodies. Protein amounts of both HSP27 and annexin II increased after UVC irradiation in nuclear fractions of UVC-resistant cells. Cells transfected with annexin II-specific siRNA were more susceptible to UVC lethality than negative control siRNA-transfected cells. There was no significant difference in cell proliferation rates between siRNA-treated cells and control cells. These findings suggest that annexin II is an HSP27-interacted protein and involved in UVC resistance in human cells, at least, the cells tested here.
