DNA Damage Response to Ionizing Radiation Defective in the Invasive Cancer Phenotype

The purpose of this study was to determine the differences in DNA damage responses (DDRs) between immortalized normal prostate epithelial cells (PrEC) and prostate cancer cells (DU145). To study the DDRs, two protein markers were used. The first, phosphorylated KRAB Associated Protein 1 (pKAP-1) activates target genes involved in the DDR. The second, phosphorylated H2AX (pH2AX) is required for the assembly of damaged chromatin as well as for activation of checkpoints proteins. Ionizing Radiation (IR) was used to induce DNA damage. Flow Cytometry and Western Blot analyses were used to measure the levels of the proteins when exposed to various doses of IR (0, 2, 4, 8 Gy). In both cell lines, there was a clear dose dependent increase in pH2AX and pKAP-1, however error bars showed that with the pH2AX protein, only the later doses showed significant differences. Fluorescence staining was used to examine the localization of the proteins. Both cell lines showed punctate staining of pH2AX in the nuclei at 8Gy and no staining in unirradiated cells. With pKAP-1, the PrEC cells had punctate staining in the nuclei with 8Gy of radiation whereas the DU145 cells had a diffuse pattern in the nucle