RADIATION SENSITIZATION APPLIED TO CANCER RADIOBIOLOGY

我们计划继续我们目前对影响和 硫醇耗竭在细胞放射增敏中的作用机制 以及新陈代谢在没有和存在各种电子的情况下- 仿生剂，包括氧气。对使用硫醇的兴趣 耗竭作为一种潜在的临床低氧治疗方法 细胞数量增加是因为(1)实验室证据 证明硫醇耗竭会增加缺氧细胞 放射增敏和(2)疾病患者5- 羟脯氨酸尿症可耐受细胞内慢性低浓度 谷胱甘肽(GSH)硫醇水平在多大程度上充当 细胞放射反应的决定因素以及是否有放射反应 需要特定类别的硫醇的专一性，例如GSH， 总的非蛋白质硫醇或蛋白质硫醇，将被评估。 将使用各种化学耗尽剂作用于 组织培养中的哺乳动物细胞和人类细胞的遗传学 缺乏谷胱甘肽合成酶，因此在某些情况下已知有 降低谷胱甘肽水平。实验将被设计来评估 至少三个可能会改变的过程的贡献 硫醇被操纵时的辐射反应：化学、生化 和酶修复。对于要检查的方面，请参阅 敏化作用与：联合的有效性有关 作用于不同细胞内硫醇库的化合物， 硫醇在细胞内定位的重要性及其相关性 硫醇相关酶活性与辐射敏感性之间的关系，以及 硫醇对辐射后酶修复过程的影响 耗尽。此外，我们建议确定如何组合 硫醇耗尽剂和电子亲和剂可以增强 在不增加细胞毒性的情况下进行放射反应。在这 连接后，将实验数据与各种模型进行比较 对辐射作用进行测试的适用性和预测性 这些型号的容量。最后，我们将研究它们之间的关系 DNA损伤，特别是双链断裂与细胞 杀戮，以及细胞内硫醇的操纵控制 水平可以改变这种关系。这些产品的主要终端 研究将使用细胞进行细胞存活，即克隆形成能力 包括中国仓鼠卵巢细胞(CHO)、中国仓鼠 肺成纤维细胞(V79)和人肺癌A549细胞 AS-GSH合成酶缺陷型人成纤维细胞及其正常 对口单位。待检测的DNA损伤包括单项和 双链断裂，用碱性和中性过滤器测定 分别采用洗脱技术。将测量硫醇水平 使用成熟的方法。我们认为这项工作提出了 之所以意义重大，是因为除了它对 目前关于放射生物学机制的知识，它将有助于 提供应用所必需的基本放射生物学知识 硫醇操作在临床上的应用，其中一些已经 开始了。

We plan to continue our current studies on the effects and mechanisms of action of thiol depletion on cell radiosensitization and metabolism in the absence and presence of various electron- affinic agents, including oxygen. Interest in the use of thiol depletion as a potential clinical approach for dealing with hypoxic cells has been heightened because (1) laboratory evidence demonstrates that thiol depletion increases hypoxic cell radiosensitization and (2) patients with the disease 5- oxoprolinuria can tolerate chronically low intracellular glutathione (GSH). The degree to which thiol levels serve as a determinant of cell radioresponse and whether radioresponse requires specificity for a particular class of thiol, e.g., GSH, total non-protein thiols or proteins thiols, will be assessed. Use will be made of various chemical depleting agents acting on mammalian cells in tissue culture and of human cells genetically deficient in GSH synthetase, and thus known, in some cases, to have decreased GSH levels. Experiments will be designed to assess the contributions of at least three processes which may alter radioresponse when thiols are manipulated: chemical, biochemical and enzymatic repair. For aspects to be examined with regard to sensitization are related to: the effectiveness of combinations of compounds acting on different intracellular thiol pools, the importance of intracellular localization of thiols, the correlation between thiol-related enzyme activity and radiosensitivity, and the alteration of post-irradiation enzymatic repair processes by thiol depletion. Further, we propose to determine how combinations of thiol-depleting agents and electron-affinic agents can enhance radioresponse without increasing cell toxicity. In this connection, experimental data will be compared with various models of radiation action to test the applicability and predictive capacity of those models. Finally, we will study the relationship between DNA damage, especially double-strand breaks, and cell killing, and whether manipulative control of intracellular thiol levels can alter that relationship. The major endpoint for these studies will be cell survival, i.e., clonogenicity, using cell lines including Chinese hamster ovary cells (CHO), Chinese hamster lung fibroblasts (V79) and human lung carcinoma A549 cells, as well as GSH-synthetase-deficient human fibroblasts and their normal counterparts. The DNA damage to be measured includes single and double-strand breaks, determined using alkaline and neutral filter elution techniques respectively. Thiol levels will be measured using well-established methods. We believe that the work proposed is significant because, in addition to its potential impact on current knowledge of radiobiological mechanisms, it will help provide basic radiobiological knowledge essential for application of thiol manipulation in the clinic, some of which has already begun.