ANTICANCER DRUG RESISTANCE BY BCL2 ONCOGENE EXPRESSION

本研究的目的是确定由于bcl-2癌基因表达导致的癌症化疗药物耐药的机制。 Bcl-2是调节细胞凋亡的蛋白质家族的原型成员，在许多不同类型的人类癌症中异常表达，包括淋巴瘤以及乳腺、前列腺、肺和结肠的肿瘤。Bcl-2阻断细胞凋亡，从而导致肿瘤细胞对诸如放疗和化疗的抗癌治疗产生抗性。 bcl-2阻断细胞凋亡的机制尚不清楚，但它似乎与bcl-2在细胞内膜（包括线粒体膜和核膜）中的定位有关。 在这些位点，bcl-2似乎调节参与凋亡级联反应的物质或因子的转运。 然而，bcl-2的其他活性也需要解释。 其他观察结果表明，bcl-2可能在细胞的抗氧化途径中发挥作用，特别是那些涉及谷胱甘肽（GSH）等硫醇的途径。 与该应用相关的进展已经证明，bcl-2在核膜水平上通过主动转运将GSH重新分配到细胞核中，在细胞核中它明显抑制进行凋亡过程的蛋白酶。 如果这个模型被进一步验证，表明GSH是阻断细胞凋亡的物质，那么可以设计出克服这种阻断并恢复抗癌药物敏感性的策略。在本应用中，将解决以下问题，以测试该模型的预测。 1. bcl-2蛋白家族的其他成员是否也参与GSH代谢？ 2. bcl-2表达是否也改变线粒体中的GSH水平？ 3. 新开发的靶向bcl-2功能或GSH本身的策略是否会使肿瘤细胞对抗癌药物敏感？ 4. 肿瘤细胞中bcl-2的表达是否受NF-κ B转录因子的调控？ 所使用的方法包括定量测量细胞内池中的GSH和通过免疫印迹分析测定bcl-2和其他蛋白的相对表达。 细胞凋亡和克隆形成生存期将用作细胞对化疗药物应答的相关终点。

The goal of this research is to define the mechanism responsible for the resistance to cancer chemotherapy agents due to expression of the bcl-2 oncogene. Bcl-2, the prototypic member of a family of proteins that regulate apoptosis, is abnormally expressed in many different types of human cancer including lymphomas, and tumors of the breast, prostate, lung and colon. Bcl-2 blocks apoptosis and, thus, causes tumor cells to become resistant to anticancer treatments such as radiotherapy and chemotherapy. The mechanism by which bcl-2 blocks apoptosis is not understood, but it appears to be related to bcl-2's localization in intracellular membranes including mitochondrial membranes and the nuclear envelope. At these sites, bcl-2 appears to regulate the transport of substances or factors involved in the apoptotic cascade. However, additional activities of bcl-2 also require explanation. Other observations indicate that bcl-2 may have a role in the cell's antioxidant pathways, specifically those involving thiols such as glutathione (GSH). Progress related to this application has demonstrated that bcl-2, at the level of the nuclear envelope, redistributes GSH into the nucleus by active transport where it apparently inhibits the proteases that carry out the apoptotic process. If this model is further validated to indicate that GSH is the substance blocking apoptosis, strategies may be devised to overcome such a block and restore anticancer drug-sensitivity. In this application, the following questions that serve to test predictions of this model, will be addressed. 1. Are other members of the bcl-2 family of proteins also involved in GSH metabolism? 2. Does bcl-2-expression also alter GSH levels in mitochondria? 3. Will newly developed strategies that target bcl-2 function or GSH itself act to sensitize tumor cells to anticancer drugs? 4. Is bcl-2 expression in tumor cells under the control of the NF-kappaB transcription factor? The methods to be used include quantitative measurements of GSH in intracellular pools and determinations of relative expression of bcl-2 and other proteins by immunoblot analysis. Apoptosis and clonogenic survival will be used as relevant endpoints for cell response to chemotherapy agents.