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Initial aspects of the molecular mechanisms of cell-substrate interactions were studied under physiologically relevant shear conditions using a parallel-plate flow chamber. The adhesion process was dissected into the initial events of arrest and stabilization using the methodology developed in this study employing video microscopy coupled to digital image processing on a SPARC 2 workstation. The cell-substrate interactions were studied using human melanoma (i.e. parental (MeWo) and two variants selected for resistance to wheat germ agglutinin cytotoxicity (3s5 and 70w)) and Chinese hamster ovary (CHO) cells expressing different levels of the fibronectin integrin, $\alpha\sb5\beta\sb1$, on their surface. These cells allowed investigation of the roles of transglutaminase (TGase) and the integrin surface receptors in the arrest and stabilization events. TGase inhibitors, monodanyslcadaverine and INO-3178, used in the studies with the melanoma cells showed inhibition of the stabilization event with no measurable effect on the arrest event when adhesion to immobilized fibronectin was investigated under flow conditions. Besides fibronectin, other immobilized proteins were evaluated using the melanoma cells such as laminin, vitronectin, type I and type IV collagen, von Willebrand factor, wheat germ agglutinin (WGA), and Peptite-2000. The results showed the arrest and stabilization events are not necessarily mediated to the same degree by the same molecular components. For example, arrest to WGA was very favorable relative to arrest to fibronectin but stabilization was very poor. Some substrates such as human laminin showed both favorable arrest and stabilization behavior relative to fibronectin. Polyclonal antibodies to the fibronectin and vitronectin integrin receptors on the melanoma cells were used to investigate the role of these integrins in the arrest and stabilization events. It appeared that both receptors are involved to some degree in the arrest and stabilization events to both substrates. The adhesion studies of the CHO cells confirmed these findings that increased fibronectin integrin enhanced arrest and stabilization to both fibronectin and vitronectin. In summary, these results suggest that the stabilization of cells to immobilized proteins is in part attributed to transglutaminase cross-linking integrin receptors on the surface of tumor cells to proteins via lysine-glutamine linkages. |
