EFFECT OF CALMODULIN INHIBITORS ON ADRIAMYCIN RESISTANCE

这项建议的基本主题是研究 钙调素抑制剂在体内外调节血管紧张素转换酶中的作用 阿霉素的细胞毒性反应及交叉耐药 渐进性阿霉素耐药肿瘤模型。肿瘤模型 要研究的将包括对父母敏感和渐进性的 小鼠白血病P388和L1210的阿霉素耐药变异体 和B16-BL6小鼠黑色素瘤。即将推出的钙调蛋白抑制剂 被评价的是三氟拉嗪和N-(4-氨基丁基)-5-氯-2- 萘磺酰胺(W-13)的亲油性显著降低， 更具特异性的钙调蛋白抑制剂。敏感而敏感的 渐进性耐药肿瘤的特征将确定 钙调蛋白抑制剂对：(A)分布的影响 从细胞匀浆中获得的亚细胞级分中的阿霉素 差示离心法测定阿霉素的含量 (B)高效液相色谱(HPLC)； 阿霉素诱导DNA蛋白质交联，DNA单双链 链断裂，以及使用 碱性和中性洗脱技术。咖啡因的作用 阿霉素在阿霉素存在和缺失时的细胞毒性研究 钙调蛋白抑制剂将专注于确定改变 在阿霉素蓄积过程中，细胞内游离钙的变化 浓度、对DNA的损伤和细胞毒性。蜂窝 阿霉素水平将用高效液相色谱法和激光流动法进行测定 细胞学。细胞内游离钙水平将通过 钙离子荧光指示剂Quin-2及其细胞毒性 琼脂集落试验。渐进性阿霉素的关系 电阻、交叉电阻特性和调制 钙调素抑制剂与长春新碱等抗肿瘤药物的联合作用 生物碱、表鬼臼毒素、蒽环类和非蒽环类 具有与DNA结合的可变亲和力的试剂将在 活着。体内抗肿瘤作用将通过体积测量来确定 P388和L1210白血病患者生存期延长的变化 以及肿瘤体积的变化和/或肺组织的缩小 转移性B16-BL6黑色素瘤。取得抵抗力 阿霉素在体内将用B16-BL6黑色素瘤进行评估。 耐药细胞的转移特性将基于 实验性和自发性转移瘤的形成，以及 阿霉素对vLTRO体内和体内抗肿瘤作用的反应 将用软琼脂集落试验测定并在肿瘤中还原 负担分别为。长远而言，拟议的研究应 帮助我们了解收购的分子基础和 阿霉素耐药的表达及其可能的作用 钙调素抑制剂在调节阿霉素细胞毒性中的作用。

The basic theme of this proposal is to study the effect of calmodulin inhibitors in vitro and in vivo in modulating the cytotoxic response to adriamycin and cross-resistant drugs in progressively adriamycin-resistant tumor models. The tumor models to be studied will include the parent-sensitive and progressively adriamycin-resistant variants of the P388 and L1210 mouse leukemia and B16-BL6 mouse melanoma. The calmodulin inhibitors to be evaluated are trifluoperazine, and N-(4-aminobutyl)-5-chloro-2- naphthalenesulfonamide (W-13) a significantly less lipophilic and "more specific" inhibitor of calmodulin. The sensitive and progressively resistant tumors will be characterized to determine the effect of calmodulin inhibitors on: (a) distribution of adriamycin in sub-cellular fractions obtained from cell homogenates by differential centrifugation and quantification of adriamycin levels by high performance liquid chromatography (HPLC); and (b) adriamycin induced DNA protein cross links, DNA single and double strand breaks, and the repair/rejoining of these lesions using the technique of alkaline and neutral elution. The effects of caffeine on adriamycin cytotoxicity in the absence and presence of calmodulin inhibitors will be focused on determining alterations in adriamycin accumulation, changes in cellular free calcium concentrations, damage to DNA and cytotoxicity. Cellular adrlamycin levels wlll be measured by HPLC and also by laser flow cytometry. Cellular free calcium levels will be determined with the fluorescent calcium indicator Quin-2, and cytotoxicity by soft- agar colony assay. The relationship between progressive adriamycin resistance, cross resistance characteristics, and the modulating effect of calmodulin inhibitors with antltumor agents such as vinca alkaloids, epipodophyllotoxins, anthracycline and non-anthracycline agents with variable affinity to bind to DNA will be evaluated in vivo. Antitumor effects in vivo will be determined bv measuring changes in prolongation of life span with P388 and L1210 leukemia, and changes in tumor volume and/or reduction of pulmonary metastases with B16-BL6 melanoma. Acquisition of resistance to adrlamycin in vivo will be evaluated with the B16-BL6 melanoma. Metastatic characteristics of resistant cells will be based on formation of experimental and spontaneous metastases, and the response to antltumor effects of adriamycin in vltro and in vivo will be determined by soft-agar colony assay and reductlon in tumor burden respectively. The proposed studies should in the long term help us understand the molecular basis for the acquisition and expression of resistance to adriamycin and the potential role of calmodulin inhibitors in modulating adriamycin cytotoxicity.