Nagao M, Shima H, Nakayasu M, Sugimura T
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Mutat Res. 1995 Dec;333(1-2):173-9. doi: 10.1016/0027-5107(95)00143-3.
Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.
最近，人们发现了许多强效的蛋白质丝氨酸/苏氨酸磷酸酶（PPs）抑制剂。其中一些在小鼠皮肤两步致癌试验和大鼠肝脏中期试验中已被证明是肿瘤促进剂。在这些抑制剂中，冈田酸（OA）能选择性抑制PP2A，因此有人提议用它来促进对这种磷酸酶生物学作用的分析。OA对体外转化显示出双峰效应，除了这种表观遗传变化外，还会诱导明显的基因变化。用OA处理超过1周会使NIH 3T3转化细胞不可逆地变平，转染基因丢失。已知它还能诱导中国仓鼠肺细胞产生抗白喉毒素突变，并在中国仓鼠卵巢细胞和人淋巴细胞中诱导姐妹染色单体交换（SCEs）。为了分析蛋白质磷酸酶在基因稳定性中的作用，我们分离出了OA抗性突变体。已证明它们在PP2Aα催化亚基中有一个突变，其中半胱氨酸269被甘氨酸取代；并且证明该区域与OA相互作用。重组突变蛋白对OA的抗性比野生型高约4至9倍。虽然CHO细胞的OA抗性突变体表达高水平的P-糖蛋白，但提示抑制PP2A本身会导致SCE诱导。然而，已知对OA敏感的PP分子种类数量仍在增加，并且我们已经分离出一种新型OA敏感PP的cDNA。我们的研究表明，仅使用OA无法阐明PP2A的作用这一事实至关重要。
