Mechanisms of specific expression of glutathione transferase gene during hepatocarcinogenesis

肝癌发生过程中谷胱甘肽转移酶基因的特异性表达机制

基本信息

批准号：
03670138
负责人：
IMAGAWA Masayoshi
金额：
$ 1.28万
依托单位：
Osaka University (1992)The University of Tokyo (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Rat glutathione transferase P (GST-P) is expressed at low levels in normal liver but becomes highly expressed in hyperplastic nodules and in hepatocellular carcinomas during chemical hepatocarcinogenesis. To understand the regulation mechanisms of this gene, we characterized the 5'-flanking region and found that GST-P gene is regulated by at least two elements: one a strong enhancer (GPEI) and the other a silencer. GPEI is composed of two TREs (TPA-responsive element) arranged palindromically with a 3bp spacing. Interestingly, GPEI is active in embryonic carcinoma F9 cells and in C127 cells, both of which are known to have little AP-1 activity. Evidence is now accumulating that GPEI is trans-activated not only by AP-1 (c-jun/c-fos complex) as usual TRE but also by a novel factor. We have also identified a negative fragment at 400bp upstream from the cap site. This fragment functions in an orientation and position independent manner, suggesting that it is acting as a silencer (negative … More enhancer). There are several cis-elements in this region and at least three trans-acting factors bind to these elements. Partially purified SF-A (Silencer Factor A) binds to several regions in this silencer, and likely plays an important role on negative regulation of this gene. Another factor SF-B (Silencer Factor B) has been cloned by Southwestern technique.For mechanisms of specific expression of GST-P, categorically, two possibilities may be considered; one is that the GST-P gene is activated because it is located closely to a putative hepato-oncogene. Second is that the GST-P gene is not linked to the hepato-oncogene but shares some trans-activator (or repressor) with it. To discriminate these possibilities, we carried out carcinogenesis experiments using transgenic rats harboring the bacterial chloramphenicol acetyltransferase (CAT) reporter gene ligated to the upstream regulatory sequence (-2900 to +59) of the GST-P gene. In each of three independent lines tested, liver foci and nodules produced by chemical carcinogens (Solt-Farber procedure) were found to express high levels CAT activity by both CAT assay using liver cytosol and immunohistochemical study, while normal liver cells did not express any CAT activity. These results unequivocally demonstrate that the GST-P gene is trans-activated locus-independently during rat hepatocarcinogenesis. Less

大鼠谷胱甘肽转移酶P（GST-P）在正常肝脏中以低水平表达，但在化学肝癌发生过程中在增生结节和肝细胞癌中高度表达。为了了解该基因的调节机制，我们表征了5'Fancing区域，发现GST-P基因至少受到两个元素的调节：一个是一个强增强剂（GPEI），另一个是消音器。 GPEI由两个TRE（TPA响应元件）组成，并以3BP间距为单位。有趣的是，GPEI活跃于胚胎癌F9细胞和C127细胞中，这两种细胞都没有AP-1活性。现在，有证据表明，GPEI不仅像往常一样被AP-1（C-Jun/c-Fos复合物）反式激活，而且还通过新的因素进行了激活。我们还从CAP位点上游的400bp鉴定了一个负片段。该碎片以方向和位置独立的方式起作用，表明它充当消音器（负…更多增强子）。该区域中有几个顺式元素，至少三个反式作用因子与这些元素结合。部分纯化的SF-A（消音器因子A）与该消音器中的几个区域结合，并且可能在该基因的负调控中起重要作用。西南技术将SF-B（消音器B）的另一个因子（消音因子B）克隆。对于GST-P的特定表达机制，可以明确地考虑两种可能性。一种是激活GST-P基因，因为它与假定的肝癌基因紧密相关。其次是GST-P基因与肝癌无关，而是与之共享一些反式激活剂（或阻遏物）。为了区分这些可能性，我们使用携带细菌氯霉素乙酰基转移酶（CAT）报告基因基因与上游调节序列（-2900至+59）的转基因大鼠进行了致癌实验。在测试的三个独立线中，发现由化学致癌物产生的肝灶和结节（Solt-Farber手术）通过使用肝细胞质和免疫组织化学研究来表达高水平的CAT活性，而正常肝细胞没有表达任何CAT活性。这些结果明确地表明，在大鼠肝癌发生期间，GST-P基因独立于反式激活基因座。较少的