Phorbol ester induced rapid attachment and spreading of melanoma cells and the role of extracellular matrix proteins

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Abstract
Phorbol 12-myristate 13-acetate (PMA) is a tumour promotor that acts as a potent protein kinase C (PKC) activator that has significant effects on tumour cell attachment and spreading. We tested whether these effects of PMA may be observed in human melanoma cells, and whether a specific response to extracellular matrix proteins may be mediated by shifts in the expression of β1 integrins. We used cell attachment assays, video time lapse cell spreading assays, flow cytometry, function blocking monoclonal antibodies (MAbs) and PKC inhibitor Calphostin C to address these questions. We established that PMA induces a rapid and temporary enhancement of cell attachment and spreading which was not accompanied by a significant change in the expression of β1 integrins. Spreading of melanoma cells that were not stimulated with PMA could be significantly blocked with a function blocking MAb (clone P4C10) against the common β1 integrin subunit. The spreading and attachment of the PMA treated cells was also significantly reduced, but less so, after MAb treatment. The PMA enhanced cell attachment and spreading could be effectively blocked by RGD sequences and PKC inhibitor. Taken together, our data indicate that PMA induces a rapid and temporary ECM-dependent enhancement of melanoma cell attachment and spreading, and that the response to ECM components appears not to be due to significant shifts in β1 integrin expression, but rather to activation of β1 integrins.