EFFECT OF CALMODULIN INHIBITORS ON ADRIAMYCIN RESISTANCE

这项建议的基本主题是研究钙调素的作用 控制阿霉素细胞毒性反应的细胞靶点抑制剂 和交叉耐药药物在进行性阿霉素耐药肿瘤中的作用 模型 待表征的肿瘤模型将包括 L1210的亲本敏感和进行性阿霉素耐药变异体 小鼠白血病、B16-BL 6小鼠黑色素瘤和HL-60人早幼粒细胞 白血病 待评价的钙调素抑制剂为三氟拉嗪， 和1，3-二氢-1-[1-[（4-甲基-4H，6H-吡咯并[1，2-a][4，1]-苯并氧氮杂卓-4-基）-N-（4-甲基-4H，6H-吡咯并[1，2-a] 甲基]-4-哌啶基]-2H-苯并咪唑-2-酮（1：1）马来酸盐（CGS 9343 B）a 不抑制蛋白激酶的选择性钙调蛋白抑制剂 活动 敏感的和进行性阿霉素耐药的肿瘤将 表征以确定：（a）细胞蓄积/保留，以及 钙调蛋白抑制剂的亚细胞分布在缺乏或 使用放射性标记药物检测阿霉素、依托泊苷和长春新碱的存在 和/或高效液相色谱法（HPLC）;和（B） 核因子调控阿霉素或足叶乙甙诱导的DNA损伤 在不存在或存在以下情况下，这些病变的损伤和修复/重新连接 单独使用分离的细胞核或重建的细胞核的钙调蛋白抑制剂 与膜和/或胞质溶胶部分，通过碱性 洗脱 此外，阿霉素或依托泊苷诱导的DNA损伤基于 与拓扑异构酶II的170 kDa和180 kDa同种型的相互作用将 通过条带消减-免疫印迹技术测定。 的改变 P-糖蛋白和拓扑异构酶II的磷酸化及其调节 钙调素抑制剂将由十二烷基钠 免疫沉淀物的硫酸盐-聚丙烯酰胺凝胶电泳。 的 P-糖蛋白和拓扑异构酶II的磷酸化免疫沉淀物 将进一步表征以确定磷酸化的氨基酸， 薄层电泳和磷酸肽消化后， 金黄色葡萄球菌V8蛋白酶 纯化的拓扑异构酶II将是 通过快速蛋白从敏感和阿霉素抗性细胞中分离 液相色谱法和表征的催化活性的基础上P4 使用独特的3'末端解结，药物诱导的DNA蛋白质交联 标记的SV-40 DNA和钙/钙调蛋白依赖性磷酸化 蛋白激酶11。 钙调素抑制剂在调节 抗性和抗性表型的出现将表征为 选择对阿霉素加， 三氟拉嗪，通过评价细胞毒性反应，诱导DNA损伤 和磷酸化的推定的目标，即P-糖蛋白和 拓扑异构酶II，在不存在或存在钙调蛋白抑制剂的情况下。 的 从药理学和生物化学的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抗性的表达。 拓扑异构酶II或长春花生物碱和钙调素的潜在作用 抑制剂调节细胞毒性的细胞效应。

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.