THE EFFECTS OF BREFELDIN-A (BFA) ON CELL-CYCLE PROGRESSION INVOLVING THE MODULATION OF THE RETINOBLASTOMA PROTEIN (PRB) IN PC-3 PROSTATE-CANCER CELLS

Purpose: To investigate the effects of brefeldin A (BFA) on the growth of the androgen-independent human prostate cancer PC-3 cells, focusin g on cell cycle regulation. Materials and Methods: BFA is a fungal mac rocyclic lactone with an antiviral activity. PC-3 cells were cultured with various concentrations of BFA for indicated times and cell growth was monitored at each time point. Cell cycle analysis was performed t o explore the mechanism of PEA-induced growth inhibition. To further i nvestigate the cell cycle regulation, cell cycle-controlling factors, such as the retinoblastoma gene product (pRB) and its regulatory compo nents cdk2, cdk4, and cyclin D-1, were analyzed by Western immunoblots . Results: BFA was a potent growth inhibitor at a concentration of 30 ng./ml., resulting in a >70% reduction in cell number at 3 days. Cell cycle analysis revealed a cell arrest in the G(1) to S phase transitio n. Western blots further showed that BFA induced dephosphorylation of pRB accompanied by down regulation of cdk2, cdk4, and cyclin D-1 expre ssion. The extended pRB dephosphorylation in control cell lysates was also observed by the addition of BFA-treated lysates, but was prevente d by the inclusion of phosphatase inhibitors in assay mixtures. Conclu sion: These results suggest that BFA may be a potent cell cycle modula tor, which post-translationally regulates pRB phosphorylation possibly by down-regulating cdk2, cdk4, and cyclin D-1 and/or by up-regulating a phosphatase(s) capable of dephosphorylating pRB. Thus, BFA-induced growth inhibition in PC-3 cells appears to be at least partially due t o the modulation of a pRB-mediated growth pathway.