THE RETINOBLASTOMA GENE-PRODUCT INHIBITS TGF-BETA-1 INDUCED APOPTOSISIN PRIMARY RAT HEPATOCYTES AND HUMAN HUH-7 HEPATOMA-CELLS

Transforming growth factor-beta 1 (TGF-beta 1) can induce rapid growth arrest and apoptosis in hepatic cells. Its growth suppressive effects appear to be linked to decreased phosphorylation of the protein produ ct of the retinoblastoma gene, pRb. To characterize the role of pRb in apoptosis, we examined endogenous retinoblastoma gene (Rb) expression following treatment with TGF-beta 1, okadaic acid, or antisense Rb S- oligonucleotides in cultured primary rat hepatocytes acid human hepato ma HuH-7 cells. We also investigated the effects on apoptosis of Rb ov erexpression following transfection with vectors containing wild-type Rb in HuH-7 cells. Our results indicated that transfection with Rb ant isense S-oligonucleotides blocked the expression of pRb in cultured pr imary hepatocytes and induced apoptosis. Treatment of HuH-7 cells with TGF-beta 1 inhibited expression and phosphorylation of pRb, and also induced apoptosis. Furthermore, 93% of viable preapoptotic cells were arrested in the G(1) phase of the cell cycle. Incubation with the phos phatase inhibitor okadaic acid maintained pRb in its phosphorylated st ate, and resulted in significant apoptosis. Overexpression of wild-typ e Rb inhibited TGF-beta 1 induced apoptosis in HuH-7 cells. In contras t, overexpression of transcription factor E2F-1, a known target for th e activity of pRb, caused significant apoptosis. However, coexpression of Rb suppressed E2F-1 induced apoptosis in HuH-7 cells. Our results suggest that inhibition of pRb expression is associated with hepatocyt e apoptosis. Furthermore, E2F-1 appears to be a target in the pathway through which pRb modulates the apoptotic threshold in hepatic cells. Finally, the data suggest that these cells exit the cell cycle during the G(1) phase before progressing into apoptosis and pRb may be a nega tive regulator of this process.