ROLE OF DNA REPAIR IN CISPLATIN RESISTANCE

顺铂(CDDP)是一种双功能dna结合型抗肿瘤药物。 在临床癌症中起主要作用。CDDP结合和去除的动力学研究 提示DNA修复与其细胞毒作用有关。抵抗力 对人类癌细胞中的cDDP是多因素的，并增强了DNA修复 可能就是这样一种机制。脱氧核糖核酸修复和脱氧核糖核酸酶的抑制剂 DNA拓扑异构酶II(TOP II)(不一定是通过相同的机制)， 通过以下途径增强顺铂耐药人癌细胞系的细胞毒作用 增加cDDP DNA链间交联度(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 抗药性突变体)细胞系将被利用。的组件类型 CDDP与基因组DNA的加合物将通过无焰原子进行检测：总cDDP 用碱洗脱法和链内交联法测定吸收光谱 用单抗进行酶联免疫吸附试验。 全基因组修复可能是一个不敏感的指标，因为活跃的基因可能 优先维修。转录活跃的基因中的ISC修复 (二氢叶酸还原酶DHFR)将与非活性(B-珠蛋白)进行比较 S和R细胞中的基因。异丙酚的形成和去除效果的比较 活性基因和非活性基因以及细胞系之间的基因将由Southern 转移，杂交分析到cdna探针。 对DNA-CDDP加合物的耐受性可能与去除一样重要； 因此，功能基因的mRNA转录将被检测并 与从同一基因获得的DNA修复数据进行比较。抄写： 用Northern或Dot方法检测cDDP暴露后dhfr基因 S细胞株和R细胞系的免疫印迹。 Nb等顶级Il活化剂对增敏作用的研究 应阐明TOP II抑制cDDP加合物的作用 活性基因和非活性基因的形成和修复以及在 抄写。 总体而言，这些研究将使我们更好地了解 DNA修复途径在细胞中的作用--对cDDP的反应及其机制 CDDP耐药。TOP-Il抑制细胞周期的分子后果 CDDP-DNA加合物的修复可能为深入了解DDP-DNA加合物的作用机制 CDDP抗性。烷化剂/cDDP对DNA修复的调节作用 治疗可能提供一种新的癌症治疗方法和克服 临床对烷基化剂的耐药性。

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.