PARENT SESSION

Cell and Tissue Signaling

(PP253) Cyclin E modulates radiation sensitivity by altering the function of cell cycle checkpoints and DNA repair in glioblastoma cells.

Aguilera, Joe³, Chou, David², Milligan, Jamie², Zhang, Lingxiao³, Lee, Guk Haeng⁵, Yoo, Hyung Jun⁴, Pardo, Francisco^2,3, Rajni, Sethi^*,1, ³ Moores Cancer Center, San Diego, CA, USA² Department of Radiation, San Diego, CA, USA⁵ Department of Otolaryngology, Korea, South Korea⁴ Department of Radiation Oncology, Seoul, South Korea¹ School of Medicine, San Diego, CA, USA

ABSTRACT- Purpose/Objective: The deregulation of molecular checkpoints in the cell cycle is associated with a number of cancers. Intrinsic radiation sensitivity varies with cell cycle phase, with S phase being the most radioresistant. Regulatory events at G1 through S may be important determinants of radiation sensitivity by altering checkpoint function as well as DNA repair mechanisms. Through their ability to alter radiation sensitivity, molecular determinants of the G1/S transition may thus be important targets of future cancer therapies. Materials/Methods: To establish which cell cycle determinants enhance radiation sensitivity, a tetracycline-inducible system was used to overexpress cyclins E, D1, and A in rat embryo cells. Cyclin upregulation was confirmed with Western Blot analysis. Cells underwent standard clonogenic assays after Cs-137 irradiation. Following the results of this experiment, a glioblastoma multiforme (GBM) line was transduced with a recombinant adenovirus containing human cyclin E cDNA. To investigate the effect of cyclin E on DNA repair, radiation-induced double-strand DNA breaks were quantified using pulsed-field gel electrophoresis one hour after irradiation. To investigate cell kinetics as a modulator of radiation toxicity, parallel flasks cultured in identical conditions and cell cycle distribution was followed over a 96-hour period after transduction using FACS analysis. Results: Clonogenic assays revealed that cells overexpressing cyclin E, as contrasted with those selectively inducing cyclins D1 or A, are significantly more sensitive to ionizing radiation (p<0.05, two-tailed test, Do, MID). Pulsed-field gel electrophoresis demonstrated significantly decreased DNA repair at doses of 30-90 Gy in cyclin E populations as compared to control populations (p<0.05, two-tailed test, beta). FACS analysis demonstrated significantly altered cell cycle distributions over a 96-hour period following transduction. Cyclin E populations had more cells in S phase and fewer in G1 phase (p<0.05, two-tailed test), while G2 distributions were not significantly different. Conclusions: Cyclin E transduction, resulting in cyclin E overexpression, sensitizes GBM and rat embryo cells to ionizing radiation. High levels of cyclin E pushed cells from G1 into S phase. The chromosomally unstable cells exposed to ionizing radiation at later cell cycle phases continue to divide, thus abrogating cell cycle checkpoint control and altering clonogenicity. Additionally, the data suggest that cyclin E overexpression interferes with DNA double-strand break repair mechanisms regulating clonogenic radiation survival. In conclusion, the data support the hypothesis that cyclin E overexpression alters the radiation sensitivity phenotype by both DNA repair and checkpoint control mechanisms.

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