Cell death in irradiated prostate epithelial cells: Role of apoptotic and clonogenic cell kill

Dose-escalated conformal radiotherapy is increasingly being used to radically treat prostate cancer with encouraging results and minimal long-term toxicity, yet little is known regarding the response of normal or malignant prostate cells to ionizing radiation (IR). To clarify the basis for cell killing during prostate cancer radiotherapy, we determined the IR-induced expression of several apoptotic- (bax, bcl-2, survivin and PARP) and G1-cell cycle checkpoint- (p53 and p21^WAF1/Cip1) related proteins, in both normal (PrEC-epithelial and PrSC-stromal) and malignant (LNCaP, DU-145 and PC-3; all epithelial) prostate cells. For these experiments, we chose doses ranging from 2 to 10 Gy, to be representative of the 1.8-2 Gy daily clinical fractions given during curative radiotherapy and the 8-10 Gy single doses given in palliative radiotherapy. We observed that IR-induced bax and P21^WAF1/Cip1 protein expression were attenuated selectively in normal stromal and epithelial cell cultures, yet maintained their p53-dependency in malignant cell lines. For each cell culture, we also determined total apoptotic and overall radiation cell kill using a short-term nuclear morphologic assay and a long-term clonogenic survival assay, respectively. Clonogenic survival, as measured by the surviving fraction at 2 Gy (SF2), ranged from 0.05 (PrEC) to 0.55 (DU-145), suggesting that malignant prostate cells are more radioresistant than normal prostate cells, for this series. IR-induced apoptotic cell kill was minimal (less than 6% cell after a dose of 10 Gy at times of 24-96 h) and was not dose-dependent. Furthermore, apoptotic kill was not correlated with either molecular apoptotic response or clonogenic cell kill. Using a flow cytometric proliferation assay with the PrSC (stromal) and DU-145 (epithelial) representative cultures, we observed that a senescent-like phenotype (SLP) emerges within a sub-population of cells post-irradiation that is non-clonogenic. Terminal growth arrest was dose-responsive at 96 h following irradiation and associated with long-term expression of both p21^WAF1/Cip1 and p16^INK4a genes. Future strategies for prostate radiotherapy prediction or novel treatments should additionally focus on terminal growth arrest as an important endpoint in prostate cancer therapy.