ROLE OF DNA REPAIR IN CISPLATIN RESISTANCE

顺铂（cDDP）是一种双功能DNA结合抑制剂， 在临床癌症中的重要作用。 cDDP结合和清除的动力学 表明DNA修复参与其细胞毒性作用。 电阻 cDDP在人类癌细胞中的作用是多因素的， 可能就是这样一种机制。新生霉素（NB），一种DNA修复抑制剂， DNA拓扑异构酶II（顶部II）（不一定通过相同的机制）， 增强cDDP耐药人癌细胞系中cDDP的细胞毒性， 增加cDDP DNA链间交联（ISC）。 我们建议研究DNA修复在cDDP耐药中的作用 在人类癌细胞中。SCC 25（S）和SCC 25/CP（R）（稳定的30倍 药物抗性突变体）细胞系。的组件类型 将测定基因组DNA的cDDP加合物：通过无火焰原子吸收法测定总cDDP 吸收光谱法，碱性洗脱和链内交联法ISC 通过ELISA使用单克隆抗体。 全基因组修复可能是一个不敏感的指标，因为活性基因可能 优先修复。转录活跃基因中的ISC修复 （二氢叶酸还原酶DHFR）将与无活性（B-珠蛋白）进行比较 S和R细胞中的基因。间充质干细胞形成和去除的比较 活性和非活性基因以及细胞系之间的关系将通过Southern杂交进行研究。 转移，并对cDNA探针进行杂交分析。 DNA-CDDP加合物的耐受性可能与去除对耐药性同样重要; 因此，将测定来自功能基因的mRNA转录， 与从相同基因获得的DNA修复数据进行比较。转录 将通过北方或斑点杂交法测量cDDP暴露后DHFR基因 在S和R细胞系中进行印迹。 NB和其他顶级Il活性剂增强敏感性的作用 的R细胞应阐明顶II抑制cDDP加合物的作用 形成和修复的活性和非活性基因和水平 转录。 总的来说，这些研究将有助于更好地了解 DNA修复途径在顺铂细胞应答中的作用及其机制 cDDP耐药。抑制top Il对 cDDP-DNA加合物的修复可能提供了对 cDDP耐药。烷化剂/cDDP后DNA修复的调节 治疗可能为癌症治疗提供一种新的方法， 烷化剂耐药的临床表现。

Cisplatin (cDDP) is a bifunctional DNA binding antineoplastic agent with a major role in clinical cancer. The kinetics of cDDP binding and removal suggest that DNA repair is involved in its cytotoxic effects. Resistance to cDDP in human cancer cells is multifactorial, and enhanced DNA repair may be one such mechanism. Novobiocin (NB), an inhibitor of DNA repair and of DNA topoisomerase II (top II) (not necessarily by the same mechanism), enhances cDDP cytotoxicity in a cDDP resistant human carcinoma line by increasing cDDP DNA interstrand crosslinks (ISC). We propose to investigate the role of DNA repair in drug resistance to cDDP in human cancer cells. The SCC 25 (S) and SCC 25/CP (R) (a stable 30-fold drug resistant mutant) cell lines will be utilized. The component types of cDDP adducts to genomic DNA will be assayed: total cDDP by flameless atomic absorption spectroscopy, ISC by alkaline elution and intrastrand crosslinks by Elisa employing monoclonal antibodies. Total genomic repair may be an insensitive indicator since active genes may be preferentially repaired. ISC repair in an actively transcribed gene (Dihydrofolate reductase DHFR) will be compared to an inactive (B-globin) gene in S and R cells. Comparisons of ISC formation and removal between active and inactive genes and between cell lines will be made by Southern transfer, and hybridization analysis to cDNA probes. Tolerance of DNA-CDDP adducts may be as important to resistance as removal; therefore mRNA transcription from a functional gene will be assayed and compared with DNA repair data obtained from the same gene. Transcription of the DHFR gene after cDDP exposure will be measured by Northern or dot blotting in the S and R cell lines. The effects of NB and other top Il active agents to enhance the sensitivity of R cells should elucidate the role of top II inhibition on cDDP adduct formation and repair in active and inactive genes and on levels of transcription. In the aggregate these studies will lead to a better understanding of the role of DNA repair pathways in the cellular.response to cDDP and mechanisms of cDDP resistance. The molecular consequences of inhibition of top Il on the repair of cDDP-DNA adducts may provide insight into the mechanism of cDDP resistance. Modulation of DNA repair after alkylating agent/cDDP therapy may offer a novel approach to cancer therapy and in overcoming alkylating agent resistance in the clinic.