We have obtained two procaspase 3 activators (PAC-1 and sPAC-1) from our collaborators at the University of Illinois, and have obtained a panel of cell lines on which to test the compounds with and without X-irradiation. Initial experiments with a number of sarcoma cell lines showed that they did not form discrete colonies when plated at low density, indicating that the gold standard evaluation of the effects of radiation (colony formation assay) could not be applied directly. We have evaluated several other assays based on phosphorylated gamma-H2AX histone proteins to quantitate the effect of radiation in both the absence and presence of procaspase 3 activators. In addition, we are validating the use of an ATP-based assay to determine radiosensitizing activity in cell lines. This has progressed significantly, and we have begun definitive assays on the cell lines. This has revealed several promising results. We are also concurrently testing the function of apoptotic machinery in cell lines under investigation. We are currently validating these assays for each sarcoma cell line.
