EFFECT OF CALMODULIN INHIBITORS ON ADRIAMYCIN RESISTANCE

这项建议的基本主题是研究钙调蛋白的作用 调控阿霉素细胞毒性反应的细胞靶点的抑制物 渐进性阿霉素耐药肿瘤的交叉耐药研究 模特们。待鉴定的肿瘤模型将包括 L1210亲本敏感和渐进性阿霉素耐药变异体 小鼠白血病、B16-BL6小鼠黑色素瘤和HL-60人早幼粒细胞 白血病。待评估的钙调蛋白抑制剂为三氟拉嗪， 和1，3-二氢-1-[1-[(4-甲基-4H，6H-吡咯并[1，2-a][4，1]-苯并恶卓-4- Yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one(l:l)maleate(CGS9343B)a 不抑制蛋白激酶的选择性钙调蛋白抑制剂 活动。对阿霉素敏感和渐进性耐药的肿瘤 其特征是确定：(A)细胞积累/保持和 钙调素抑制剂在无菌条件下的亚细胞分布 用放射性标记药物检测阿霉素、依托泊苷和长春新碱 和/或高效液相色谱仪；和(B) 核外因子对阿霉素或依托泊苷诱导DNA的调控作用 在没有或存在这些损害的情况下，这些损害的损害和修复/重新连接 单独使用或重组核使用钙调蛋白抑制剂 膜和/或胞浆组分，通过碱性技术 洗脱。此外，阿霉素或依托泊苷引起的DNA损伤基于 与拓扑异构酶II 170 kDa和180 kDa亚型的相互作用 用条带耗竭-免疫印迹技术测定。中的更改 P-糖蛋白和拓扑异构酶II的磷酸化及其调控 钙调素抑制剂将由十二烷基钠测定 免疫沉淀物的硫酸盐-聚丙烯酰胺凝胶电泳法。这个 P-糖蛋白和拓扑异构酶II的磷酸化免疫沉淀物 将进一步表征以通过以下方式确定磷酸化氨基酸 薄层电泳法与酶消化后的磷酸肽 金黄色葡萄球菌V8蛋白酶。纯化的拓扑异构酶II将被 用快速蛋白从敏感和阿霉素耐药细胞中分离 基于P4的高效液相色谱及其催化活性表征 解开，药物诱导的DNA蛋白利用独特的3‘端进行交联 标记SV-40 DNA与钙/钙调素依赖性的磷酸化 蛋白激酶11.钙调素抑制物在细胞周期调控中的作用 抗性和抗性表型的出现将被表征为 肿瘤细胞被选为对阿霉素+的抗药性组合， 三氟拉嗪，通过评估细胞毒反应，诱导DNA损伤 和假定的靶分子的磷酸化，即。P-糖蛋白和 拓扑异构酶II，在没有或存在钙调蛋白抑制剂的情况下。这个 从药理学和生化角度提出的研究应该 从长远来看，帮助我们了解收购涉及的目标 以及对阿霉素和其他抑制剂的耐药性表达 拓扑异构酶II或长春花碱与钙调蛋白的潜在作用 控制细胞毒性的调节细胞效应的抑制剂。

The basic theme of this proposal is to study the effect of calmodulin inhibitors on cellular targets governing a cytotoxic response to adriamycin and cross-resistant drugs in progressively adriamycin-resistant tumor models. The tumor models to be characterized will include the parent-sensitive and progressively adriamycin-resistant variants of L1210 mouse leukemia, B16-BL6 mouse melanoma and HL-60 human promyelocytic leukemia. The calmodulin inhibitors to be evaluated are trifluoperazine, and 1,3-dihydro-1-[1-[(4-methyl-4H, 6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4- yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one(l:l)maleate (CGS9343B) a selective calmodulin inhibitor which does not inhibit protein kinase activity. The sensitive and progressively adriamycin-resistant tumors will be characterized to determine: (a) the cellular accumulation/retention and subcellular distribution of calmodulin inhibitors in the absence or presence of adriamycin, etoposide and vincristine using radiolabelled drugs and/or high performance liquid chromatography (HPLC); and (b) the role of extranuclear factors in governing adriamycin or etoposide induced DNA damage and repair/rejoining of these lesions in the absence or presence of calmodulin Inhibitors using isolated nuclei alone or nuclei reconstituted with membrane and/or cytosol fractions, by the technique of alkaline elution. Further, adriamycin or etoposide induced DNA damage based on Interaction with the 170 kDa and 180 kDa isoforms of topoisomerase II will be determined by band depletion-immunoblotting technique. Alterations in phosphorylation of P-glycoprotein and topoisomerase II and its modulation by calmodulin inhibitors will be determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. The phosphorylated immunoprecipitates of P-glycoprotein and topoisomerase II will be further characterized to determine phosphorylated amino acids by thin layer electrophoresis and phosphopeptides following digestion with Staphylococcus aureus V8 protease. Purified topoisomerase II will be isolated from sensitive and adriamycin-resistant cells by fast protein liquid chromatography and characterized for catalytic activity based on P4 unknotting, drug induced DNA protein cross linking using uniquely 3' end labelled SV-40 DNA and phosphorylation by calcium/calmodulin dependent protein kinase 11. The role of calmodulin inhibitors in modulation of resistance and emergence of a resistant phenotype will be characterized in tumor cells selected for resistance to a combination of adriamycin plus, trifluoperazine, by evaluating cytotoxic response, induction of DNA damage and phosphorylation of putative targets viz. P-glycoprotein and topoisomerase II, in the absence or presence of calmodulin inhibitors. The proposed studies from a pharmacological and biochemical standpoint should in the long term help us understand the targets involved in the acquisition and expression of resistance to adriamycin and other inhibitors of topoisomerase II, or vinca alkaloids and the potential role of calmodulin inhibitors in modulating cellular effects governing cytotoxicity.