Role of protein kinase C isoforms in locomotion of Walker 256 carcinosarcoma cells

Treatment with low (nanomolar) concentrations of phorbol-12-myristate-13-ac etate (PMA) for 5 to 30 min suppresses locomotion of Walker 256 carcinosarc oma cells, suggesting that activation of protein kinase C (PKC) is a stop s ignal for tumor cell locomotion. We have compared the effects of PMA on cel l shape and motility with down-regulation of specific PKC isoforms. Using s pecific antibodies, we show that Walker carcinosarcoma cells express PKC is oforms alpha, beta I, beta II, gamma, lambda, mu, eta and zeta. Short-term incubation with PMA induced a marked shift of isoforms alpha, beta I, beta II, gamma and eta to the particulate fraction. Long-term incubation with PM A (0.1 mu M, 6 hr) resulted in significant reduction of expression of conve ntional PKCs alpha, beta I, beta II and gamma and of the novel PKC eta to 1 0% to 26% of controls. Down-regulation of PKC alpha, beta I and beta II by long-term incubation with PMA was reversible after removal of PMA, whereas that of isoforms gamma and eta was not. The motile properties of cells afte r down-regulation of PKC isoforms were investigated. Concomitant with down- regulation of PKC isoforms, long-term incubation of cells with PMA resulted in recovery of the polar shape and the ability to migrate. Motility and po larized shape of the down-regulated cells were no longer susceptible to sho rt-term treatment with PMA, showing that active PKC is indeed responsible f or the inhibitory effects of PMA. Effects of long-term incubation with PMA on cell shape and motility were reversible. Our findings strongly suggest t hat PKCs alpha, beta I and beta II activated by PMA are involved in stoppin g Walker carcinosarcoma cell locomotion. Int. J. Cancer 81:255-261, 1999. ( C) 1999 Wiley-Liss, Inc.