Search

Protein phosphatase inhibitor-1 plays an important role in the regulation of glycogen metabolism through inhibition of protein phosphatase-1 activity, and it has been implicated in the regulation of cell growth. Using real-time quantitative RT-PCR, we studied the mRNA expression of inhibitor-1 in hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine, and in a non-tumorigenic liver cell line (C1I), that stores glycogen in excess during early passages. In late passages, glycogen is gradually lost concomitant with cell transformation. Our in vitro model included a tumorigenic subline of C1I cells that was obtained by chemically-induced neoplastic transformation using N-methyl-N'-nitro-N-nitrosoguanidine (C1Ict), and does not store glycogen, as well as Morris hepatoma 3924A (MH3924A) cells. We found that in hepatocellular carcinomas, in the late glycogen-poor passages (C1I(late)), and in the tumorigenic subline (C1Ict) of C1I cells, and in MH3924A cells the mRNA expression of inhibitor-1 is significantly increased. This increase in expression varied from 15 to 290-fold of that observed in normal liver. In contrast, in the early glycogen-storing passage of C1I cells (C1I(early)) the level of inhibitor-1 mRNA was found to be slightly less than that of normal liver. Inhibitor-1 mRNA levels correlated with the degree of differentiation of HCCs. These results indicate that the expression of inhibitor-1 mRNA is tightly linked to tumor progression and to the process of liver cell transformation in vitro and is inversely correlated with the glycogen content of the cell.

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.