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

Abstract

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 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.

WTp53 induction does not override MTp53 chemoresistance and radioresistance due to gain-of-function in lung cancer cells

“…At no point did we see any evidence of apoptosis in this system, suggesting that perhaps H1299 cells are not easily predisposed to p53-induced apoptosis, which is a consistent observation in solid epithelial tumor cell lines (48) and also consistent with previous studies using the murine orthologue (44). Although this article was being written, another report using the p53 A138V mutant in human pancreatic cancer cell lines also showed not only a lack of apoptosis but also a lack of induction of Bax (68).…”

“…Induction of DNA Damage and Clonogenic Assays DNA DSBs were induced by cellular g-irradiation using a Gammacell irradiator (Noridon International) with a Cs 137 source as described previously (48). Cells were prepared for UV irradiation by removing all media and a PBS wash from tissue culture plates and then irradiated with 10 J/m 2 using a 254 nm Stratalinker UV source (Stratagene).…”

Androgens induce oxidative stress and radiation resistance in prostate cancer cells though NADPH oxidase

“…Radiation survival was calculated as the plating efficiency of treated cells divided by the plating efficiency of untreated cells as previously described. 17 …”

Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

“…Clonogenic assays were performed after gas and drug/IR treatments as previously described (34). IR was used to induce DNA-dsbs.…”