B16-BL6 mouse melanoma cells cultured on fibronectin coated dishes were detached by treatment with echistatin, an RGD-containing disintegrin. Echistatin was active at micromolar concentrations and was not cytotoxic. Its effect was dose-dependent and reversible. Sequential morphological changes leading to rounding up of the cells were deterred by phase-contrast microscopy and by immuno fluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytoplasmic actin filaments were observed well before cell detachment occurred. Echistatin treatment down-regulated the phosphorylation of pp125(FAK) in fibronectin-adherent cells in a dose-and time-dependent fashion. The reduction of pp125(FAK) phosphorylation preceded cell detachment and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125(FAK) phosphorylation and that echistatin acts by inhibiting protein tyrosine kinase activity rather than activating protein tyrosine phosphatases.
Echistatin induces decrease of pp125(FAK) phosphorylation, disassembly of actin cytoskeleton and focal adhesions, and detachment of fibronectin-adherent melanoma cells
ESPOSITO, Sabrina;
1997
Abstract
B16-BL6 mouse melanoma cells cultured on fibronectin coated dishes were detached by treatment with echistatin, an RGD-containing disintegrin. Echistatin was active at micromolar concentrations and was not cytotoxic. Its effect was dose-dependent and reversible. Sequential morphological changes leading to rounding up of the cells were deterred by phase-contrast microscopy and by immuno fluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytoplasmic actin filaments were observed well before cell detachment occurred. Echistatin treatment down-regulated the phosphorylation of pp125(FAK) in fibronectin-adherent cells in a dose-and time-dependent fashion. The reduction of pp125(FAK) phosphorylation preceded cell detachment and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125(FAK) phosphorylation and that echistatin acts by inhibiting protein tyrosine kinase activity rather than activating protein tyrosine phosphatases.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
