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操作所需的基本设备，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，每次不超过10种化合物将被发送给我们进行测试。NCGC还为我们提供了50%致死剂量（LD50）的参考值，这是由他们的分析确定的。通过对细胞内ATP含量的定量分析（类似于NCGC最初使用的方法），我们证实了我们能够在LD50药物浓度下复制大约50%的细胞毒性，并提供前10种化合物。将辐射与前10种药物结合进行ATP含量测定的初步实验表明，这10种化合物中的一种可能在培养中起放射增敏剂的作用。验证性分析正在进行中，并将侧重于更标准化的定量分析，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，我们最初发现我们的原代脊索瘤细胞系UCH-1在稀疏培养时不会形成菌落。我们已经用几种方法来解决这个问题。1. 我们已经验证了基于atp的检测方法对辐射致敏的可靠报告。2. 我们已经确定了提高天然细胞系形成菌落能力的生长条件，并处于优化该技术的最后阶段。3. 市售的磷酸化γ H2AX保留测定法正在被优化，作为一种替代的测定输出。4. 每个核伽马H2AX保留测定也被使用，并已优化。我们已经开始在上述条件下测试NCGC化合物的辐射致敏性。

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.