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，但在化学致癌过程中在增生性结节和肝细胞癌中高表达。为了了解该基因的调控机制，我们对GST-P基因的5‘侧翼区进行了研究，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。GPEI由两个TPA反应元件(TRE)组成，回文排列，间隔3bp。有趣的是，GPEI在胚胎癌F9细胞和C127细胞中都很活跃，这两种细胞都被认为几乎没有AP-1的活性。现在越来越多的证据表明，GPEI不仅像往常一样被AP-1(c-jun/c-fos复合体)反式激活，而且还被一种新的因子激活。我们还在CAP位点上游的400bp处发现了一个负片段。该片段以独立于方向和位置的方式起作用，表明它起到了消音器的作用(负…更多增强剂)。该区域有几个顺式元件，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(沉默因子A)与该沉默因子中的几个区域结合，可能在该基因的负调控中发挥重要作用。另一种因子SF-B(沉默因子B)已经通过西南技术被克隆。对于GST-P特异表达的机制，绝对可以考虑两种可能性：一种是GST-P基因被激活，因为它位于一个假定的肝癌基因附近。其次，GST-P基因与肝原癌基因没有关联，但与其共享一些反式激活因子(或抑制因子)。为了区分这些可能性，我们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了致癌实验，该报告基因连接到GST-P基因的上游调控序列(-2900到+59)。在三个独立的品系中，由化学致癌物(Solt-Farber程序)产生的肝脏病灶和结节在肝细胞胞浆CAT检测和免疫组织化学研究中均表达高水平的CAT活性，而正常肝细胞不表达任何CAT活性。这些结果明确地证明了GST-P基因在大鼠肝癌发生过程中是非反式激活的。较少

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