CALMODULIN INHIBITORS EFFECT ON ADRIAMYCIN RESISTANCE

本申请的基本主题是建立机制， 拓扑异构酶II使阿霉素或依托泊苷中毒， 钙-钙调蛋白调节过程，三氟拉嗪和1-[N-O- 双（1，5-异喹啉磺酰基）-N-甲基-L-酪氨酰-4-苯基哌嗪（KN- 62)合作敏化抗性细胞。 我们假设 细胞内游离钙和拓扑异构酶II的磷酸化是 足叶乙甙诱导的DNA可切割复合物形成所需， 细胞毒 实验将使用敏感或 进行性阿霉素耐药HL-60和L1210肿瘤模型 系统. 为了检测钙调素抑制剂的增敏作用是否 依托泊苷或阿霉素依赖于游离钙，细胞将 用细胞内钙缓冲液处理， 药物蓄积、DNA可裂解复合物的形成和细胞凋亡将 被确定为参与增强 细胞毒 钙调素抑制剂对磷酸化的影响 拓扑异构酶II将在对照组或钙缓冲剂治疗组中进行评价 用[32 P]-正磷酸标记的细胞。 使用拓扑异构酶II 免疫沉淀，我们将表征，药物诱导的DNA裂解 复合物的形成由带耗尽和磷酸肽由两个 三维胰蛋白酶图谱。 药物诱导DNA损伤的稳定性 它对拓扑异构酶II磷酸化的依赖性也将 被确定。 钙调素抑制剂的细胞周期时相特异性 将在同步化的细胞群中通过确定以下来评估：（a） 药物稳定的DNA可切割复合物的形成、凋亡和 细胞毒性;和（B）拓扑异构酶II的磷酸化及其 免疫沉淀物中的肽。 从长远来看， 药理学表征的事件调节的调制 钙调蛋白抑制剂的细胞毒性应有助于理解 对临床有用的拓扑异构酶II的耐药机制 抑制剂的

The basic theme of this application is to establish mechanisms by which the topoisomerase II poisons adriamycin or etoposide and the inhibitors of calcium-calmodulin regulated processes, trifuluoperazine and 1-[N-O- bis(1,5-isoquinolinesulfony)-N-methyl-L-tyrosyl-4phenylpiperazine(KN- 62) co-operate in sensitizing resistant cells. We hypothesize that intracellular free calcium and the phosphorylation of topoisomerase II are required for etoposide induced DNA cleavable complex formation and cytotoxicity. Experiments will be carried out using sensitive or progressively adriamycin-resistant HL-60 and L1210 tumor model systems. To test whether the sensitizing effect of calmodulin inhibitors on etoposide or adriamycin is dependent on free calcium, cells wil be treated with an intracellular calcium buffer and drug accumulation, DNA cleavable complex formation and apoptosis will be determined as potential mechanisms involved in enhancing cytotoxicity. The effect of calmodulininhibitors on pohosphorylation of topoisomerasde II will be evaluated in control or calcium bufffer treated cells labelled with [32P]-orthophosphoric acid. Using topoisomerase II immunoprecipitates we will characterize, drug induced DNA cleavable complex formation by band depletion and phosphopeptides by two dimensional tryptic mapping. Stability of drug induced DNA damage and its dedpendence on the phosphorylation of topoisomerase II will also be determined. The cell cycle phase specificity of calmodulin inhibitors will be evaluated in synchronized cell populations by determining: (a) drug stablilized DNA cleavable complex formation, apoptosis and cytotoxicity; and (b) the phosphorylation of topoisomerase II and its peptides in immunoprecipitates. In the long term a ciochemical and pharmacological characteriszation of events regulating the modulation of cytotoxicity by calmodulin inhibitors should aid in the understanding of mechanisms of resistance to clinically usefule topoisomerase II inhibitors.