Regulation mechanisms of glutathione transferase gene expression 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。为了解GST-P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）。化学致癌物引起的肝病灶和结节组织中CAT活性均呈高水平表达，而正常肝细胞不表达CAT活性。这些结果表明，GST-P基因在大鼠肝癌发生过程中是反式激活位点非依赖性的。在GPEI-CAT转基因大鼠中也得到了类似的结果，表明GPEI是GST-P基因在肝癌发生过程中激活的重要顺式元件，并在帽位点上游300 bp处发现了沉默元件。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。我们纯化了沉默因子A（Silencer Factor A，SF-A），并测定了其部分氨基酸序列。有趣的是，SF-A似乎是NF 1（核因子1）的相关蛋白，NF 1是转录和DNA复制的激活因子。另一种因子SF-B（沉默因子B）已被克隆并发现与LIP（肝抑制蛋白）相同，LIP是LIP（肝激活蛋白）的竞争者。通过GAL 4 DNA结合域转染实验，我们发现LIP不仅是竞争因子，而且是直接阻遏因子。

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.We carried out carcinogenesis experiments using transgenic rats harboring chloramphenicol acetyltransferase (CAT) reporter gene with -2900 to +59of the GST-P gene. Liver foci and nodules produced by chemical carcinogens were found to express high levels CAT activity by both CAT assay and immunohistochemical study, while normal liver cells did not express any CAT activity. These results demonstrate that the GST-P gene is trans-activated locus-independently during rat hepatocarcinogenesis. Moreover, the similar results were obtained using transgenic rats carrying GPEI-CAT,indicating that GPEI is an important cis-elements for activation of GST-P gene during hepatocarcinogenesis.We have also identified a silencer element at 300bp upstream from the cap site. There are several cis-elements in this region and at least three trans-acting factors bind to these elements. We purified SF-A (Silencer Factor A) binds to several regions in this silencer, and determined the partial amino acid sequence. Interestingly, SF-A seemed to be related protein to NF1 (Nuclear Factor 1) which is activator for transcription and DNA replication. Another factor SF-B (Silencer Factor B) has been cloned and found to be same as LIP (Liver Inhibitory Protein) which is a competitor for LAP (Liver Actovator Protein). By transfection assay using GAL4 DNA binding domain, however, we found LIP is not only a competitor but a direct repressor.