EFFECT OF CALMODULIN INHIBITORS ON ADRIAMYCIN RESISTANCE

本提案的基本主题是研究 钙调蛋白抑制剂在体外和体内调节 对阿霉素和交叉耐药药物的细胞毒反应 进行性阿霉素耐药肿瘤模型。 的肿瘤模型 将包括父母的敏感性，并逐步 P388和L1210小鼠白血病的阿霉素耐药变异体 和B16-BL 6小鼠黑素瘤。 钙调蛋白抑制剂是 评价的是三氟拉嗪和N-（4-氨基丁基）-5-氯-2- 萘磺酰胺（W-13）是一种显著较低的亲脂性和 钙调蛋白的“更特异性”抑制剂。 敏感和 进行性耐药肿瘤将被表征以确定 钙调蛋白抑制剂对以下方面的影响：（a） 从细胞匀浆中获得的亚细胞级分中的阿霉素 通过差速离心和阿霉素定量 通过高效液相色谱法（HPLC）测定的水平;和（B） 阿霉素诱导的DNA蛋白质交联，DNA单链和双链 链断裂，以及使用 碱性和中性洗脱技术。 咖啡因的影响 对阿霉素细胞毒性的影响 钙调蛋白抑制剂将集中在确定改变 在阿霉素蓄积中，细胞游离钙的变化 浓度、DNA损伤和细胞毒性。 蜂窝 阿霉素水平将通过HPLC和激光流测量 细胞仪 细胞游离钙水平将通过 荧光钙指示剂Quin-2和细胞毒性软- 琼脂集落测定 进展性阿霉素 电阻、交叉电阻特性和调制 钙调素抑制剂与抗肿瘤药物如长春花的作用 生物碱、表鬼臼毒素、蒽环类和非蒽环类 与DNA结合的亲和力可变的试剂将在 vivo. 体内抗肿瘤作用将通过测量 P388和L1210白血病患者寿命延长的变化， 以及肿瘤体积的变化和/或肺肿瘤体积的减少。 B16-BL 6黑色素瘤转移。 获得耐药性 将用B16-BL 6黑色素瘤评价体内阿霉素。 耐药细胞的转移特征将基于 实验性和自发性转移的形成，以及 体外和体内对阿霉素抗肿瘤效应的反应 采用软琼脂集落法和肿瘤还原法测定 分别负担。 从长远来看，拟议的研究应 帮助我们了解收购的分子基础， 阿霉素抗性的表达及 钙调蛋白抑制剂调节阿霉素细胞毒性。

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.