In the first year since establishing the laboratory, we have acquired the basic equipment required for laboratory operation, and executed the Materials Transfer Agreements necessary for us to obtain the 27 compounds from the NCGC. Per our agreement with NCGC, no more than 10 compounds will be sent to us at a time for testing. NCGC has also provided us with the reference 50% lethal dose (LD50) values, as determined by their assays. Using quantitative assays of intracellular ATP content (similar to those used initially by NCGC), we have confirmed our ability to replicate roughly 50% cytotoxicity at LD50 drug concentrations in culture with the first 10 compounds supplied. Initial experiments combining radiation with the first 10 drugs in ATP content assays have suggested that one of the 10 compounds may act as a radiosensitizer in culture. Confirmatory assays are ongoing and will focus on more standardized quantitative assays, as described below. In other efforts to establish reliable quantitative assays of the effects of ionizing radiation on chordoma cells, we initially found that our primary chordoma cell line UCH-1 does not form colonies when sparsely cultured. We have approached this problem in several ways. 1. We have validated ATP-based assays for reliable reporting of radiation sensitization. 2. We have identified growth conditions that improve the ability of the native cell lines to form colonies, and are in the final stages of optimizing this technique. 3. Commercially available phosphorylated gamma H2AX retention assays are being optimized as an alternative assay output. 4. Per nucleus gamma H2AX retention assays are also being used and have been optimized. We have begun testing the NCGC compounds for radiation sensitization using the conditions above.
