Overexpression of regucalcin suppresses cell proliferation in cloned rat hepatoma H4‐II‐E cells: Involvement of intracellular signaling factors and cell cycle‐related genes

Abstract

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild type) and stable regucalcin/pCXN2 transfectants were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayters. The proliferation of cells was significantly suppressed in transfectants cultured for 24–72 h. The proliferation of wild-type cells was significantly inhibited when the cells were cultured for 72 h in a medium containing an inhibitor of transcriptional activity or protein synthesis. Such an effect was not seen in transfectants. The presence of various inhibitors of protein kinase including PD 98059 (10⁻⁷ or 10⁻⁶ M), dibucaine (10⁻⁶ M), wortmannin (10⁻⁸ or 10⁻⁶ M), or genistein (10⁻⁵ M) caused a significant inhibition of the proliferation of wild-type cells. These inhibitory effects were not seen in transfectants. Staurosporine (10⁻⁸ − 10⁻⁷ M) significantly inhibited the proliferation of wild-type cells and transfectants. Also, the effect of vanadate (10⁻⁵ M), an inhibitor of protein tyrosine phosphatase, or Bay K 8644 (10⁻⁶ M), an agonist of calcium entry into cells, in inhibiting the proliferation of wild-type cells was not observed in transfectants. Moreover, the proliferation of wild-type cells was significantly inhibited in the presence of roscovitine (10⁻⁷ or 10⁻⁶ M) or sulforaphane (10⁻⁷ M), which induces cell-cycle arrest. Such effect was not seen in transfectants. The inhibitory effect of sodium butyrate (8.3 × 10⁻⁴ M) on proliferation of wild-type cells was also induced in transfectants. Gene expression in hepatoma cells cultured for 72 h with 10% FBS was determined by using reverse transcription-polymerase chain reaction (RT-PCR). The expression of p21 mRNA was significantly enhanced in transfectants, while cdc2a and chk2 mRNA expression were not significantly changed. Insulin-like growth factor-I (IGF-I) mRNA expression was significantly suppressed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation that is partly mediated through various intracellular signaling-related factors, and that the effect may be partly involved in the change in p21 or IGF-I mRNA expression. The finding further supports that regucalcin plays an important role as a suppressor in the enhancement of cell proliferation.