Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by the immunosuppressants FK506 and cyclosporin A

We examined alterations in cell morphology and expression of adhesion molec ules in response to a general protein kinase inhibitor K252a treatment of n on-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell ad hesion and spreading with concomitant formation of actin stress fibers. A p rotein kinase A inhibitor KT5720 also induced cell adhesion, but the rate o f spread was slower than that seen with K252a. These adhesions were mediate d by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, an d was inhibited by monoclonal antibodies for integrins alpha(2) and beta(1) . Indirect immunofluorescence microscopic observations revealed that integr in alpha(2) and beta(1) molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxi llin increased during K252a- or KT5720-induced cell adhesion. Immunosuppres sants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion an d abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, als o inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.