Mechanisms of specific expression of glutathione transferase gene during hepatocarcinogenesis

大鼠谷胱甘肽转移酶P（GST-P）在正常肝脏中低水平表达，但在增生结节和化学性肝癌过程中的肝细胞癌中高度表达。为了了解该基因的调控机制，我们对GST-P基因的5 '侧翼区进行了分析，发现GST-P基因至少受两个元件的调控：一个是强增强子（GPEI），另一个是沉默子（silencer）。GPEI由两个间隔3bp的TPA-responsive element（TPA-responsive element）组成。有趣的是，GPEI在胚胎癌F9细胞和C127细胞中有活性，已知这两种细胞几乎没有AP-1活性。现在越来越多的证据表明，GPEI不仅像通常的TRE一样被AP-1（c-jun/c-fos复合物）反式激活，而且还被一种新的因子激活。我们还在帽位点上游400 bp处鉴定了一个阴性片段。该片段以方向和位置独立的方式发挥作用，表明它是作为沉默物（阴性 ...更多信息 增强子）。在该区域中有几个顺式元件，并且至少有三个反式作用因子与这些元件结合。部分纯化的SF-A（Silencer Factor A）与该沉默子中的几个区域结合，并且可能在该基因的负调控中起重要作用。另一个因子SF-B（Silencer Factor B）已被Southwestern技术克隆，其特异性表达的机制有两种可能：一种可能是GST-P基因被激活，因为它位于一个假定的肝癌基因附近。第二，GST-P基因与肝癌基因不相关，但与肝癌基因有一些共同的反式激活因子（或阻遏因子），为了区分这些可能性，我们使用携带细菌氯霉素乙酰转移酶（CAT）报告基因的转基因大鼠进行致癌实验，该报告基因连接到GST-P基因的上游调控序列（-2900至+59）。在每三个独立的线测试，肝病灶和结节产生的化学致癌物（Solt-Farber程序）被发现表达高水平的CAT活性的CAT测定使用肝细胞质和免疫组织化学研究，而正常肝细胞不表达任何CAT活性。这些结果明确地表明，GST-P基因是反式激活位点的非依赖性大鼠肝癌的发生。少

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