Overexpression of regucalcin modulates tumor-related gene expression in cloned rat hepatoma H4-II-E cells

Abstract

Regucalcin is a regulatory protein in intracellular signaling pathway which is related to various protein kinases and protein phosphatases in many cells. The effect of regucalcin on the expression of tumor-related genes was investigated in the cloned rat hepatoma H4-II-E cells and the hepatoma cells (transfectants) overexpressing regucalcin. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). The proliferation of hepatoma cells was significantly suppressed at 24-72 h of culture in regucalcin transfectants as compared with that of wild-type or mock-type cells. Western blot analysis showed that regucalcin was markedly expressed in transfectants. The expression of c-myc, c-fos, c-jun, Ha-ras, and p53 mRNAs was determined using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, the expression of c-myc or Ha-ras mRNAs was significantly suppressed in regucalcin transfectants. The suppression of c-myc mRNA expression in transfectants was confirmed by using Northern blot analysis; significant suppression was seen at 24, 48, or 72 h of culture in the presence of 10% FBS. Culture with 10% FBS significantly enhanced c-myc mRNA expression in the hepatoma cells (wild-type) as compared with that of 1% FBS. The enhancement was significantly abolished in the transfectants. Meanwhile, the expression of p53 mRNA in the hepatoma cells was significantly enhanced in regucalcin-overexpressing hepatoma cells. This study demonstrates that the expression of oncogene c-myc and Ha-ras mRNA in hepatoma cells overexpressing regucalcin is suppressed, and that the tumor suppression gene p53 is enhanced in the transfectants.