Genetical and toxicological studies for evaluation of clinical tests in individualities

临床试验中有两个品系的大鼠血清亮氨酸氨基肽酶活性存在差异，这种差异通过F1、F2和回交大鼠的实验得到了遗传控制。由于N末端20个氨基酸序列在人、牛和大鼠中都是保守的，所以我们在保守区域内设计了引物，利用大鼠肝脏cDNA文库进行了DNA扩增。扩增产物与预期大小一致，但与保守氨基酸序列无相似性。我们正在尝试使用其他启动子和其他cdna文库进行聚合酶链式反应。LAP在某些肿瘤细胞中的活性较高，需要锌。接下来，我们研究了细胞转化与金属硫蛋白(MT)之间的关系，金属硫蛋白被认为是锌的代谢。由于MT的表达受转录调控，因此将MT I启动子插入报告基因LacZ的上游，并将所得到的质粒导入正常细胞和包括ras、sis或neu癌基因之一的转化细胞。金属或糖皮质激素处理后，β-半乳糖苷酶活性在正常和ras转化的细胞中被诱导，在sistran转化的细胞中被诱导，而在新转化的细胞中被抑制。MT含量较高也被认为是导致对癌症化疗药物顺铂产生耐药性的原因。Ras、sis、neu转化细胞对顺铂的耐受性高于正常细胞。这些结果表明，在NE转化细胞中，MT基因的表达受ras和sis癌基因诱导的MT I启动子的调控，可能受MT II启动子或其他因素的调控。

There are two rat strains which showed different serum leucine amino-peptidase activity, one of the clinical tests, and the difference was turned to be genetically controlled by the experiments using F1 and F2 as well as back-crossed rats. Bcause N-terminal 20 amino acid sequences were available and conserved among human, bovine and rat, we made the primers within the conserved region, amplified DNA by PCR using rat liver cDNA library. PCR product showed the expected size but no sequence similarity to conserved amino acids. We are trying PCR using other promers, and also other cDNA libraries.LAP activity is higher in certain tumor cells and requires zinc. We next examined the relation between cell transformation and metallothionein (MT) which is thought to function as zinc metabolism. Because MT expression is controlled transcriptionally, MT I promoter was inserted upstream of the reporter gene (lacZ) and the resulting plasmid was introduced to normal cells as well as transformed cells including one of the ras, sis or neu oncogene. After treatmer of metal or glucocorticoid, beta-galactosidase activity was induced in normal and ras-transformed cells, superinduced in sistransformed cells, but repressed in neu-tranformed cells. Highter MT content was also thought to cause the resistance to Cisplatin which is one of cancer chemotherapeutic agents. ras, sis, neu transformed cells showed higher resistance to Cisplatin compared to normal cells. From these results, we concluded that MT gene expression was controlled by MT I promoter which was induced by ras and sis oncogene, and might be controlled by MT II promoter or other factors in ne transformed cells.