Genetical and toxicological studies for evaluation of clinical tests in individualities

用于评估个体临床测试的遗传和毒理学研究

• 批准号: 05670325
• 负责人: DOI Rikuo
• 金额: $ 1.41万
• 依托单位: Yokohama City University, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670325/
• 关键词: Individuality; Clinical test; Leucine amino-peptidase; Metallothionein; Promoter; Oncogene; Cell transformation; Cisplatin; 固体差

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.

有两个品系的大鼠显示出不同的血清亮氨酸氨基肽酶活性，临床试验之一，并通过使用F1和F2以及回交大鼠的实验，将这种差异转化为遗传控制。由于N端20个氨基酸序列在人、牛和大鼠中存在一定的保守性，我们在保守区内设计引物，利用大鼠肝脏cDNA文库进行PCR扩增。PCR产物显示预期大小，但与保守氨基酸没有序列相似性。我们正在尝试使用其他的启动子和其他的cDNA文库进行PCR。在某些肿瘤细胞中，DNA的活性更高，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代谢的功能。由于MT表达受转录控制，因此将MT I启动子插入报告基因（lacZ）的上游，并将所得质粒引入正常细胞以及包括ras、sis或neu癌基因之一的转化细胞。经金属或糖皮质激素处理后，正常细胞和ras转化细胞的β-半乳糖苷酶活性被诱导，在Sisttransformed细胞中被过度诱导，而在neu转化细胞中被抑制。较高的MT含量也被认为导致对癌症化疗药物之一顺铂的耐药性。ras、sis、neu转化细胞对顺铂的耐药性明显高于正常细胞。结果表明，在ne-transformed细胞中，MT基因的表达受MT Ⅰ启动子的调控，而MT Ⅱ启动子的调控可能与其他因素有关。