Anticancer drug resistance studies using Dictyostelium

人类肿瘤经常对许多广泛使用的化疗药物产生耐药性。对于这种抗性的分子基础，人们提出了许多不同的机制。对肿瘤细胞耐药的研究多集中于药物的作用机制，并寻找由于细胞内药物浓度改变、药物失活或损伤修复改变而产生的耐药性。因此，可能涉及细胞对个别药物的细胞毒性反应的其他途径被忽视了。我们建议使用细胞黏液霉菌Dictyostelialdiscoideum以一种无偏见的方法来识别新的分子靶点，这些靶点可以被调节来增加肿瘤细胞对化疗药物的敏感性。网盘基菌的基因和途径与人类高度保守，分子遗传学方法已经为这种生物发展得很好。我们初步建立的顺铂耐药体系共鉴定出6个基因。值得注意的是，这些药物以前都没有与顺铂相关，每一个都代表了一个潜在的治疗新靶点。本研究的目的是证明该系统的普遍实用性，并表明它可以应用于了解对其他药物的耐药性。我们重点介绍了四类破坏DNA的药物，它们通过不同的机制破坏DNA。这些包括：链内和链间交联剂、单烷基化物和氧自由基。我们将1)建立一个完整的Dictyostoma插入突变体文库，2)分离这四类药物的耐药突变株并鉴定其同源基因，以及3)测试突变株对其他药物的交叉耐药性。这些研究将确定新的耐药机制和化疗干预的新靶点，随后可以在人类细胞中进行验证。

Human tumors frequently develop resistance to many of the widely used chemotherapeutic agents. Many different mechanisms have been proposed for the molecular basis of this resistance. Studies on drug resistance in tumor cells have often focused on the mechanism of action of the drug, and looked for resistance due to altered drug concentration in the cell, different models of drug inactivation or altered damage repair. As such, other pathways that may be involved with the cellular cytotoxic response to individual drugs have been overlooked. We propose to use the cellular slime mold Dictyostelium discoideum in an unbiased approach to identify novel molecular targets that can be modulated to increase sensitivity of tumor cells to chemotherapeutic drugs. The genes and pathways of Dictyostelium are highly conserved with those of humans, and molecular genetic methods are well developed for this organism. Our preliminary system on cisplatin resistance resulted in the identification of 6 genes. Significantly, none of these had been previously associated with cisplatin, and each represents a potential new target for therapy. The goal of the present study is to demonstrate the general utility of this system and to show that it can be applied to the understanding of resistance to other drugs. We have focused on four classes of DNA damaging drugs that damage DNA by different mechanisms. These include: both intra- and inter-strand crosslinkers, monoalkylators, and oxygen radicals. We will 1) create a comprehensive Dictyostelium insertional mutant library, 2) isolate mutants resistant to drugs of each of the four classes and identify the cognate genes, and 3) test the mutants for cross-resistance to the other drugs. These studies will identify new mechanisms for drug resistance and new targets for chemotherapeutic intervention which can subsequently be validated in human cells.