Zheng B, Chambers T C, Raynor R L, Markham P N, Gebel H M, Vogler W R, Kuo J F
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
J Biol Chem. 1994 Apr 22;269(16):12332-8.
A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.
已建立一种对冈田酸（OA）具有抗性的人白血病K562细胞突变体（K562/OA200），OA是蛋白磷酸酶1和2A（PP1/PP2A）的抑制剂。在野生型细胞中，OA的细胞毒性与有丝分裂停滞以及浓度和时间依赖性的DNA片段化有关，这是细胞凋亡的一个标志。就对这些参数的影响而言，该突变体对OA的抗性高100倍。尽管几种蛋白质的合成发生了改变，但酶活性测定和免疫印迹分析表明，突变体中PP1和PP2A的水平未变。蛋白质激酶C（PKC）活性测定以及对钙依赖性（cPKC）和钙非依赖性（nPKC）亚型的免疫印迹分析显示，突变体中明显不存在nPKC-ε，而野生型中也存在的所有其他同种型（cPKC-α、cPKC-β和nPKC-ζ）在突变体中的表达量相当。Northern印迹分析证实突变体细胞中不存在PKC-ε mRNA。OA200细胞不仅对另一种PP1/PP2A抑制剂花萼海绵诱癌素A具有交叉抗性，而且对结构不相关的抗癌药物（如长春碱和紫杉醇）也具有交叉抗性，此外，在蛋白质和mRNA水平上均过表达维拉帕米敏感的药物泵P-糖蛋白。然而，该突变体对所测试的几种PKC抑制剂（包括心脏毒素、蜂毒肽、星形孢菌素和一种烷基磷脂）没有交叉抗性。在亚毒性浓度下，心脏毒素可使OA200细胞中长春碱的细胞毒性增强6倍。这些发现表明，多药耐药表型可由传统抗癌药物以外的细胞毒性剂诱导产生，表明多药耐药的发生不一定与cPKC活性增加有关，并鉴定出某些具有作为耐药调节剂潜力的PKC抑制剂。
