Promoter Activity of Hepatocyte Growth Factor Receptor in Hepatocytes Responding to Cellular Stress

• Main Authors: Feng-Peng Chang, 張豐鵬
• Other Authors: Chih-Sheng Lin
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/86693117147567446159

碩士 === 國立交通大學 === 生物科技系所 === 94 === Liver damage is the most common organ toxicity caused by drugs and is a major health problem. Many drugs have been withdrawn from clinical use because of being recognized as potential hepatotoxins. The evaluation of the hepatic effects of a chemical before human exposure consequently represents a critical step and in vitro hepatotoxicity test is an important procedure of the pilot and high-throughput assay system. It has been demonstrated that hepatocyte growth factor/c-Met (hepatocyte growth factor receptor) signaling pathway is required for efficient liver regeneration and repair. Therefore, the aim of the present study is to develop an experimental in vitro model to study c-Met expression for hepatocyte poisoning. In this study, human c-Met promoter-Luc (luciferase) and c-Met promoter-EGFP (enhanced green fluorescent protein) were constructed by cloning 322-, 649-, and 952-bp of the 5�S-flanking sequence of human c-Met into the promoterless Luc and EGFP-containing plasmid vector. We made use of a Rattus norvegicus normal liver cell line (clone 9) that expresses a recombinant luciferase or EGFP gene fused to c-Met promoter gene. Consequently, turning on the promoter gene causes the reporter gene to be turned on. Activation or suppression of the promoter gene by chemicals lead to production of reporter signal that ultimately generate fluorescence or luminescence. By transfection of 0.9, 0.6, 0.3 met-Luc/EGFP, we have clearly suggested that potential regulatory elements conserved between human and rat are likely to be actively involved in transcriptional regulation of the c-Met gene. To understand the hepatotoxic effect on transcriptional regulation of c-Met expression in clone 9 cells, the clone 9 cells were treated with LC50 concentration of acetaminophen, D-galactosamine, and thioacetamide. The results have shown that 20 mM acetaminophen, 60 mM D-galactosamine and 150 mM thioacetamine significantly reduced the c-Met promoter activity in the clone 9 cells harboring 0.9 met-Luc. Our results suggest that after hepatotoxic drugs-treated cells, downregulation of c-Met gene is crucial to regenerate because it decreases the activity of this receptor/ligand system with a lower targeting HGF action. In addition, the HGP expression was not detectable in the clone 9 cells treated with acetaminophen or not by the RT-PCR in this study. However, it is known that other organs can supply this protein to the liver through blood, which may account for its high levels in plasma of hepatitis patients. Therefore, it is reasonable to suggest that the unresponsiveness of hepatotoxic drugs-treated cells to the regeneration-promoting action of HGF may be due not to the lack of HGF, but rather to the lack of expression in the liver of the c-Met protooncogene coding for the HGF receptor. Our study suggests that regardless of the level of liver HGF, c-Met protooncogene seems the most active modulator of liver cell proliferation, and its induction might be a critical factor in determining the site specificity of the receptor/ligand system and might be a useful prognostic marker for evaluating liver poisoning. Furthermore past studies have shown that c-Met gene is overexpressed in a variety of neoplastic tissues such as carcinomas of the gastrointestinal tract, melanomas, and sarcomas. Experimental evidence suggests that overexpression of the c-Met gene is mainly due to aberrant transcriptional regulation. In this study we found that carcinogenic compounds such as MNNG and EMS significantly increaseed the c-met promoter activity in the clone 9 cells harboring 0.9 met-Luc. Our observation is in complete agreement with a past study that c-Met oncogen was activated after mutagen treatment in vitro [Park et al., 1986]. Hence we believe that this system can also be used to screen carcinogens. In the present study, we have developed luciferase and EGFP-base assays for the rapid screening of compounds possessing hepatotoxic or carcinogenic properties. This cell-based functional assay could provide a platform for identifying agents with potential hepatotoxic and carcinogenic activity. We hope that the results presented here will serve to accelerate the use of living cells in screens for drug discovery. The ultimate goal of in vitro experiments is to generate the type of scientific information needed to identify compounds that are potentially hepatotoxic or carcinogenic properties. For this purpose, both the design of the experiments and the interpretation of the results are critical.