TRANSGENIC CELL MODELING OF ALKYLATING AGENT RESISTANCE

描述：总体目标是定义以下各项的相对贡献 四种关键代谢酶在决定疗效、毒性和 细胞耐药机制决定白血病的治疗效果 用环磷酰胺(CPA)及其类似物治疗。调查人员 先前的研究表明，乙醛在V79中的过度表达 脱氢酶(ALDH)-1或-3通过以下途径诱导对CPA类似物的抗性 减少DNA链间交联度；由于中间体的氧化 羟基磷酰胺(ALDO)为失活的羧基磷酰胺(CBP)。纯净的 ALDH-3不催化该反应，因此在特定的目标1中重组 将进行研究，以确定胞质辅助因子。有一个 对马福胺(MAF)的抗药性(6-10倍)高于 4-羟基环磷酰胺，推测是由于硫醇部分MESNA所致 从这个模拟器中释放出来的。MAF消耗谷胱甘肽(GSH)更多 慢慢地。ALDH抗性介导的抗性可能与内源或 胞外GSH或其他硫醇。具体目标2将定义范围 以及这种相互作用的机制。ALDH的相对疗效观察 CPA细胞内激活与细胞外激活的解毒作用尚不清楚。 研究人员将使用具有激活能力的ALDH转基因细胞来 测试ALDH途径将赋予更高的抗性的假设 CPA持续内源性激活条件下的等毒剂量 当Aldo以团注的形式出现时(Aim 3)，可能是由于 第二部分是谷胱甘肽的维持作用。第四，一种新的激活途径 与CPA对肺和膀胱的毒性有关，涉及 环氧合酶(COX-1或COX-2)将使用转基因细胞株进行研究 已经表达了这些同工酶。首先将对细胞进行检查以 确定它们是否以花生四烯酸依赖的方式激活CPA。如果是的话， 它们将被用作ALDH表达的受体 向量。将使用细胞毒性研究、代谢物和谷胱甘肽分析。 比较COX和ALDH在这些细胞中的表达，看看是否 不同的激活途径+/-之间存在差异 ALDH解毒作用(AIM 4)这些遗传学机制研究 已定义的模型将为不同注册会计师的动态提供新的见解 新陈代谢途径。

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.