ANTAGONISTIC ACTIONS OF PHORBOL ESTER IN MAMMALIAN G(0)-]G(1)-]S CELL-CYCLE TRANSITION

We have developed a protocol that reveals two antagonistic effects of phorbol-12-myristate-12-acetate (PMA) on the G(0)-->G(1)-->S transitio n of mammalian cell cycle. Balb-3T3 (Clone A31) cells arrested in G(0) by serum starvation can be stimulated to traverse the G(0) phase and initiate DNA synthesis 12 h later by a 2-h pulse with PMA. In contrast with this early stimulatory effect, PMA has an inhibitory effect when presented to the cells during the last 6 h of G(1). PMA is able to in hibit DNA synthesis initiation irrespective of the triggering agent, i .e., serum, fibroblast growth factor, epidermal growth factor, platele t-derived growth factor, or PMA itself (presented as an early pulse). We have established that the critical period for the PMA inhibitory ef fect is between 6 and 8 h after cell stimulation, This dual effect of PMA is not a peculiarity of Balb-3T3 (clone A31) cells because it is a lso observed with other fibroblastic cell lines, namely, SWISS 3T3, NI L 8, and RAT 1, and also with the epithelial Y-1 adrenocortical cell l ine. Treatment with PMA for 0.5 or 2 h activates protein kinase C (PKC ) in Balb-3T3-A31 cells, but is not sufficient to down-regulate the en zyme because a second 30-min PMA pulse applied between 6 and 6.5 h act ivates PKC again, On the other hand, a continuous 6.5-h PMA treatment causes PKC down-regulation; therefore, the inhibitory effect of PMA co uld be mediated by PKC. Growth factor early response proto-oncogenes c -myc, c-fos, and c-jun are induced transiently by both early and late PMA pulses, suggesting that these genes are not involved in the PMA in hibitory effect.