TRANSGENIC CELL MODELING OF ALKYLATING AGENT RESISTANCE

描述：总体目标是定义以下方面的相对贡献： 四种关键的代谢酶在决定疗效，毒性和 细胞耐药机制决定了 环磷酰胺（CPA）及其类似物治疗。 调查人员 先前的研究表明，醛在V79中的过表达 脱氢酶（ALDH）-1或-3导致对CPA类似物的抗性， 减少DNA链间交联;由于中间体的氧化 醛磷酰胺（ALDO）与无活性的羧基磷酰胺（CBP）。 纯化 ALDH-3不催化反应，因此在特异性目的1重构中 将进行研究以鉴定胞质辅因子。 有一个 对马磷酰胺（MAF）的耐药性高于（6-10倍） 4-羟基环磷酰胺，这可能是由于巯基部分MESNA 从这个模拟物中释放出来。 MAF消耗谷胱甘肽（GSH）更多 慢慢 ALDH抗性介导的抗性可能与细胞内或细胞外的抗ALDH抗体协同作用。 细胞外GSH或其它硫醇。 具体目标2将定义 这种相互作用的机制。 ALDH的相对疗效 CPA活化的细胞内与细胞外的解毒作用尚不清楚。 研究者将使用具有激活能力的ALDH转染细胞， 测试ALDH途径将赋予更高抗性的假设， 连续内源性激活CPA条件下的等毒性剂量 与ALDO作为推注（Aim 3）时相比，Cyt P450可能是由于 GSH维持作用的一部分。 第四，一种新的激活途径 与CPA对肺和膀胱的毒性有关，涉及通过 将使用转染的细胞系研究环氧合酶（考克斯-1或考克斯-2 已经表达了这些同工酶。 首先检查细胞， 确定它们是否以花生四烯酸依赖的方式激活CPA。 如果是这样， 它们将被用作ALDH表达转染的受体 向量。 将使用细胞毒性研究以及代谢物和GSH分析 比较考克斯和ALDH表达在这些细胞中的作用， 不同激活途径之间存在差异+/- 解毒ALDH（目的4）这些机制的研究，在遗传 定义的模型将提供新的见解，不同的CPA的动态 代谢途径

DESCRIPTION: The overall goal is to define the relative contributions of four key metabolic enzymes in determining the efficacy, toxicity and cellular resistance mechanisms the determine the therapeutic outcome of treatment with cyclophosphamide (CPA) and it analogs. The investigato'rs previous studies have shown that overexpression in V79 of aldehyde dehydrogenase (ALDH) -1 or -3 leads to resistance to CPA analogs by decreasing DNA interstrand crosslinks; due to oxidation of the intermediate aldophosphamide (ALDO) to the inactive carboxyphosphamide (CBP). Purified ALDH-3 does not catalyze the reaction, thus in Specific Aim 1 reconstitution studies will be carried out to identify cytosolic cofactors. There is a higher (6-10 fold) resistance to mafosfamide (MAF) than to 4-hydroxycyclophosphamide which is presumably due to the thiol moiety MESNA that is released from this analog. MAF depletes glutathione (GSH) more slowly. ALDH resistance-mediated resistance may synergize with intra- or extracellular GSH or other thiols. Specific Aim 2 will define the extent and mechanism of this interaction. The relative efficacy of ALDH for detoxification of CPA activated intra- versus extracellularly is unknown. The investigator will use activation-competent, ALDH-transfected cells to test the hypothesis that the ALDH pathway will confer higher resistance at equitoxic doses under conditions of continuous endogenous activation of CPA by Cyt P450 than when ALDO is presented as a bolus (Aim 3), perhaps due in part to effects of GSH maintenance. Fourth, a novel activation pathway implicated in CPA toxicity to lung and bladder, involving co-oxidation by cyclooxyenases (COX-1 or COX-2) will be studied using transfected cell lines already expressing these isozymes. The cells will be examined first to determine if they activate CPA in an arachidonatedependent manner. If so, they will be used as recipients for transfection with the ALDH expression vectors. Cytotoxicity studies and metabolite and GSH analysis will be used to compare the role of COX and ALDH expression in these cells to see if differences exist between the different activation pathways +/- detoxification by ALDH (Aim 4) These mechanistic studies in genetically defined models will provide new insights to the dynamics of different CPA metabolism pathways.