Celli Claudia M, Jaiswal Anil K
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA.
Cancer Res. 2003 Sep 15;63(18):6016-25.
Mitomycin C (MMC) is an anticancer drug that requires reductive activation to exert its toxicity. MMC is known to cross-link DNA that contributes significantly to the cytotoxicity and consequent cell death. Cytosolic NADPH:quinone oxidoreductase 1 (NQO1) and microsomal enzymes have been shown to mediate MMC-induced DNA cross-linking. However, NQO1 plays only a minor role, indicating presence of other cytosolic enzymes/proteins that contribute to this process. In this study, we have characterized a unique cytosolic activity in NQO1-null mice that catalyzed MMC-induced DNA cross-linking. This activity was cofactor independent and dicoumarol insensitive. The unique cytosolic activity was purified to homogeneity. The peptide sequencing of the purified protein identified the unique cytosolic activity as GRP58 (M(r) 58,000 glucose-regulatory protein), also known as GRp57/ER60/ERp61/HIP-70/Q2 and CPT. Immunodepletion of NQO1-null mice liver cytosol and partially purified fractions with anti-GRP58 antibody led to a complete loss of GRP58 protein and consequent significant reduction of MMC-induced DNA cross-linking. Mouse cDNA encoding GRP58 was isolated and sequenced. Chinese hamster ovary cells permanently overexpressing GRP58 showed increased MMC-induced DNA cross-linking and increased cytotoxicity on exposure to MMC. Bacterially expressed and purified GRP58 increased the MMC-induced DNA cross-linking when added to mouse cytosolic samples. A tissue array analysis indicated that GRP58 is ubiquitously expressed among mouse tissues, although at different levels. Expression analysis using matched human tumor/normal array revealed an up-regulation of GRP58 in breast, uterus, lung, and stomach tumors compared with normal tissues of similar origin.
丝裂霉素C（MMC）是一种抗癌药物，需要还原激活才能发挥其毒性。已知MMC会使DNA发生交联，这对细胞毒性及随后的细胞死亡有显著贡献。胞质NADPH：醌氧化还原酶1（NQO1）和微粒体酶已被证明可介导MMC诱导的DNA交联。然而，NQO1仅起次要作用，这表明存在其他有助于此过程的胞质酶/蛋白质。在本研究中，我们在NQO1基因敲除小鼠中鉴定了一种独特的胞质活性，该活性催化MMC诱导的DNA交联。这种活性不依赖辅因子且对双香豆素不敏感。该独特的胞质活性被纯化至同质。纯化蛋白的肽测序确定该独特的胞质活性为GRP58（分子量58,000的葡萄糖调节蛋白），也称为GRp57/ER60/ERp61/HIP-70/Q2和CPT。用抗GRP58抗体对NQO1基因敲除小鼠肝脏胞质溶胶和部分纯化级分进行免疫去除，导致GRP58蛋白完全丧失，从而使MMC诱导的DNA交联显著减少。分离并测序了编码GRP58的小鼠cDNA。永久过表达GRP58的中国仓鼠卵巢细胞在暴露于MMC时显示出MMC诱导的DNA交联增加和细胞毒性增加。当添加到小鼠胞质样品中时，细菌表达并纯化的GRP58增加了MMC诱导的DNA交联。组织阵列分析表明，GRP58在小鼠组织中普遍表达，尽管表达水平不同。使用匹配的人肿瘤/正常阵列进行的表达分析显示，与相似来源的正常组织相比，GRP58在乳腺癌、子宫癌、肺癌和胃癌中上调。
