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

Treatment with low (nanomolar) concentrations of phorbol-12-myristate-13-acetate (PMA) for 5 to 30 min suppresses locomotion of Walker 256 carcinosarcoma cells, suggesting that activation of protein kinase C (PKC) is a stop signal for tumor cell locomotion. We have compared the effects of PMA on cell shape and motility with down-regulation of specific PKC isoforms. Using specific antibodies, we show that Walker carcinosarcoma cells express PKC isoforms α, βI, βII, γ, λ, μ, η and ζ. Short-term incubation with PMA induced a marked shift of isoforms α, βI, βII, γ and η to the particulate fraction. Long-term incubation with PMA (0.1 μM, 6 hr) resulted in significant reduction of expression of conventional PKCs α, βI, βII and γ and of the novel PKC η to 10% to 26% of controls. Down-regulation of PKC α, βI and βII by long-term incubation with PMA was reversible after removal of PMA, whereas that of isoforms γ and η was not. The motile properties of cells after 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 polarized shape of the down-regulated cells were no longer susceptible to short-term treatment with PMA, showing that active PKC is indeed responsible for the inhibitory effects of PMA. Effects of long-term incubation with PMA on cell shape and motility were reversible. Our findings strongly suggest that PKCs α, βI and βII activated by PMA are involved in stopping Walker carcinosarcoma cell locomotion. Int. J. Cancer 81:255–261, 1999. © 1999 Wiley-Liss, Inc.