1992 Fiscal Year Final Research Report Summary
Mechanisms of specific expression of glutathione transferase gene during hepatocarcinogenesis
| Project/Area Number |
03670138
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Osaka University (1992)
The University of Tokyo (1991) |
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Principal Investigator |
IMAGAWA Masayoshi Osaka University, Fac. of Pharm. Sci., Associate Professor, 薬学部, 助教授 (20136823)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Glutathione transferase / Hepatocellular carcinoma / Chemical carcinogenesis / Enhancer / Silencer / Gene expression / Transgenic rats |
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| Research Abstract |
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
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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
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