Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis

BACKGROUND: The authors previously reported increased ubiquitin (Ub) immunoreactivity in hepatocellular carcinomas (HCCs) and suggested a possible correlation between changes in ubiquitinated protein levels and multistep hepatocarcinogenesis. The current study was performed to identify one of these ubiquitinated proteins (42 kDa) and to analyze the clinical significance of its accumulation. METHODS: The protein was purified using two-dimensional gel electrophoresis and identified by amino acid sequence analysis. The authors studied the expression of this protein in 101 HCCs and 23 precancerous lesions by immunohistochemical methods and in 26 HCCs by immunoblot analysis. A survival analysis was performed on patients with advanced HCC using the Kaplan-Meier method with approximate chi-square statistics for the log rank test. RESULTS: The target protein for ubiquitination was identified as glutamine synthetase (GS). Accumulation of GS was found in 19 of 49 advanced HCCs (38.8%) by immunohistochemical methods and in 9 of 16 (56.3%) by immunoblot analysis, whereas the frequency was much lower in early HCCs (12.9% and 33.3%, respectively) and precancerous lesions (4.3% by immunostaining). In the Ub immunoblot analysis of strongly GS positive specimens, an intense 42-kDa ubiquitinated band was observed. Nine of 21 (42.9%) nodule-in-nodule type HCCs showed a GS positive, high-grade component within a GS negative, low-grade component, indicating the acquisition of GS expression during progression. Among 23 patients with a single advanced HCC nodule, the relapse free survival time was significantly shorter in the GS positive group than in the GS negative group. CONCLUSIONS: The results of this study demonstrate the acquisition of GS expression during hepatocarcinogenesis and the possible regulation of GS enzyme activity by a Ub-dependent proteolytic system. Moreover, GS might play a significant role in promoting the metastatic potential of HCC.