Abstract 2673: Tetraiodothyroacetic acid (tetrac) produces an increase in the unirradiated steady-state levels of DNA double strand breaks (DSBs) and inhibition of repair of DSBs after x-irradiation in human U87MG brain tumor cells

INTRODUCTION. We measured the steady-state levels and post-x-irradiation repair of DNA DSBs by neutral comet assay in human U87MG glioblastoma cells in vitro in the absence and presence of tetrac. The results provide a mechanistic basis for our previous observations of radiosensitization and inhibition of cellular recovery after x-irradiation in murine GL261 cells by tetrac (A.H. Hercbergs et al., Cell Cycle 8:2586-2591, 2009). METHODS. U87MG cells on plastic slides were exposed to 2 nM tetrac for 1 h at 37°C. The slides were then placed on crushed ice and irradiated with doses from 0 to 75 Gy. Slides were then immersed in lysing solution at 50°C for 2 h. Slides were washed and placed in a gel electrophoresis unit. Electrophoresis was carried out at 0.66 V/cm for 25 min. Cells were stained with ethidium bromide and image analysis was carried out on an epifluorescence microscope at a total magnification of 400X, using an excitation filter of 515-535 nm and a barrier filter at 590 nm. We calculated the mean tail moment (TM) expressed in arbitrary units. TM was calculated with a CCD camera and software. Approximately 350 comets were analyzed per condition. RESULTS. We first demonstrated that human U87MG cells responded to tetrac exposure in a manner similar to that seen in murine GL261 cells. That is, we observed both radiosensitization and inhibition of radiation repair in cellular studies with both GL261 and U87MG cells. For radiosensitization. we observed a factor of approximately 2.2 in U87MG cells which is quite similar to that seen in GL261 cells. We demonstrated that DSB repair in U87 MG cells was inhibited by 2 nM tetrac by a factor of 72.5%, compared to repair observed in control U87MG cells. Again, this finding is similar to that obtained in murine glioma cells. We then demonstrated that exposure of non-irradiated U87MG cells (0 Gy) to tetrac increased the steady-state levels of DSBs, as indicated by a change in TM from 6.0 to 13.1, yielding a sensitization factor of 2.2. However, in graded single doses, tetrac did not affect the slope of the mean tail moment. Lastly, flow cytometric studies on control and tetrac-treated U87MG cells indicated these changes were not due to alteration in the cell cycle distribution of cells. CONCLUSIONS. These studies provide a linkage between cellular results, i.e., radiosensitization and inhibition of repair in fractionated repair studies, and biochemical endpoints that include increased numbers of DSBs in cells in the unirradiated state and decreased repair of DSBs in fractionated radiation experiments in tetrac-exposed cells. These studies suggest additional studies of possible inhibition of the activity of ATM kinase in tetrac-treated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2673. doi:10.1158/1538-7445.AM2011-2673