Sensitization of chordoma cell lines to ionizing radiation

在实验室成立的第一年，我们已经获得了实验室运行所需的基本设备，并签署了从NCGC获得27种化合物所需的材料转让协议。根据我们与NCGC的协议，一次将不会有超过10种化合物被送到我们进行测试。NCGC还向我们提供了由他们的检测确定的参考50%致死剂量(LD50)值。使用细胞内ATP含量的定量分析(类似于NCGC最初使用的那些)，我们已经证实了我们有能力在提供前10种化合物的情况下，在LD50药物浓度的培养中复制大约50%的细胞毒性。最初的实验将辐射与ATP含量测定中的前10种药物相结合，表明这10种化合物中的一种可能在培养中起辐射增敏剂的作用。验证性分析正在进行中，将重点放在更标准化的定量分析上，如下所述。在建立电离辐射对脊索瘤细胞影响的可靠定量分析的其他努力中，我们发现我们的原代脊索瘤细胞系UCH-1在稀疏培养时不形成集落。因此，它在其天然状态下不适合用于克隆形成试验。我们最近获得了一个衍生细胞系UCH-1N，并证明了它在培养中形成集落的能力。除了在UCH-1N细胞中使用NCGC化合物的初步实验外，我们还在筛选UCH-1N细胞在倍增时间、代谢活性和标志性蛋白表达方面的任何差异。商业上可用的磷酸化伽马H_2AX保留分析已经按照2010财年的预算订购，预计将在不久的将来到达。

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 have discovered that our primary chordoma cell line UCH-1 does not form colonies when sparsely cultured. Therefore, it is not suitable in its native state for colony formation assays. We have recently obtained a derivative cell line UCH-1N and have demonstrated its ability to form colonies in culture. In tandem with initial experiments using NCGC compounds in UCH-1N cells, we are also screening UCH-1N cells for any differences in doubling time, metabolic activity, and hallmark protein expression. Commercially available phosphorylated gamma H2AX retention assays have been ordered with the FY2010 budget and are expected to arrive in the near future.