Radiosensitization by post-irradiation treatment with an Hsp90 inhibitor, 17-DMAG, in the p53 mutant pancreatic cancer cell line, MiaPaca2

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The 90 kDa heat shock protein (Hsp90) is one of the most abundant molecular chaperones in mammalian cells. In cancer cells, Hsp90 plays a critical role in the stabilization and regulation of the activity of numerous client proteins related to oncogenesis. Previously, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin(17-DMAG) has been shown to enhance cancer cell radiosensitivity when delivered prior to irradiation (IR).This effect may be due to degradation of a number of client proteins such as Raf1, Wee1, ErbB-2 and Akt or by interfering with DNA double strand break (DSB) repair after IR, potentially through inhibition of ATM or DNA-PK function. In this study, we determined the effect on tumor cell radiosensitivity of 17-DMAG delivered 6h after IR. MiaPaca2 cells were exposed in vitro to 50 nM 17-DMAG 6h after irradiation. The addition of 17-DMAG to MiaPaca2 cells 6h after irradiation resulted in sustained increased foci of phosphorylated histone H2AX at 24h after irradiation compared to cells treated with radiation alone. This correlated with reduced clonogenic survival with post-radiation treatment with 17-DMAG compared to that observed with radiation alone or 17-DMAG alone. These results indicate a failure of DSBs repair in 17-DMAG post-treated cells. We hypothesized HR machinery for DSBs would be more efficient than NHEJ at 6h after RT based on the observed cell cycle arrest in late G2 phase. Cell cycle analysis at the 6h timepoint after irradiation in MiaPaca cells revealed an increased number of cells in late S and G2 phase. In order to investigate the hypothesis that impaired HR was responsible for the decreased clonogenic survival in cells treated with 17-DMAG 6h after IR, we investigated the protein levels of Rad 51, a central molecule of HR machinery. The protein level of Rad 51 was not dramatically decreased in 17-DMAG post-treated cells compared to irradiated cells; however, numbers of Rad 51 foci were reduced by 17-DMAG post-treatment in irradiated MiaPaca cells. Our data suggest that the inhibition of HR repair is an additional mechanism through which 17-DMAG can enhance tumor cell radiosensitivity.