Effect of protein kinase C inhibitors on invasiveness of human melanoma clones expressing different levels of protein kinase C isoenzymes.

Abstract

The involvement of protein kinase C (PKC) in the mechanism of chemotaxis and invasiveness of human melanoma has been studied in 6 clones of 665/2 cell line characterized by a different integrin profile, differentiation grade and in vitro invasive ability. The levels of total protein kinase C activity revealed a direct correlation with the chemotactic and invasive ability of these clones. Protein kinase C inhibitors, sphingosine and staurosporine, reduced chemotaxis and invasiveness of the highly invasive clone 2/60, while 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) was ineffective. Immunofluorescence analysis revealed high levels of protein kinase C alpha in clone 2/60, while the less invasive clone 2/21 expressed low levels of protein kinase C alpha and beta, but surprisingly appreciable levels of protein kinase C gamma. Downregulation with phorbol 12-myristate 13-acetate (TPA) did not affect invasiveness of clone 2/60 unless the compound was present during the assay. H7 strongly increased invasiveness of clone 2/21 and was able to reverse the inhibitory effect of TPA on clone 2/60. Preliminary experiments showed higher levels of diacylglycerol in clones with lower protein kinase C, suggesting a constitutive downregulation of the enzyme in low invasive clones. Our results support a role for protein kinase C in the invasion process, but point out the complexity of the mechanism which might involve the proteolytic fragment of the enzyme, protein kinase M.