6533b86efe1ef96bd12cc732

RESEARCH PRODUCT

Focal adhesions and assessment of cytotoxicity

subject

description

Focal adhesions are highly ordered assemblies of transmembrane receptors, extracellular matrix proteins, and a large number of cytoplasmic proteins, including structural proteins, as well as tyrosine kinases, phosphatases, and their substrates. They are now accepted as a prime component of signal transduction. Because focal adhesions also play an important role in cell morphology and migration, it can be argued that their presence is indicative of healthy cells. This has been the reason for several research groups to conclude that biomaterials sustaining focal adhesion assembly are biocompatible. In this study we demonstrate that cells under cytotoxic stress may still be able to retain their focal adhesions. Human umbilical vein endothelial cells at passage 2 were exposed to nickel and zinc ion solutions ranging from 1 to 0.01 mM for 4 and 24 h. Cells were seeded on fibronectin precoated glass slides or in tissue culture quality 96-well plates. MTT conversion with 1 and 0.5 mM nickel and zinc was strongly depressed, indicating that these concentrations are cytotoxic. Proliferative activity was also affected by these concentrations. Cells exposed to zinc typically retracted and detached from the surface, whereas cells exposed to nickel remained on the surface without signs of retraction. Nevertheless, cells exposed to nickel were impaired to reach confluency, which was determined by cadherin-5 expression. All these data indicate that nickel ions at a sufficient concentration influence cells in a cytotoxic way. Despite this apparent cytotoxicity, focal adhesion distribution as visualized by immunofluorescence staining of vinculin was not affected. With zinc the morphological changes were accompanied by apparent fusion of focal adhesions during retraction and finally dissolution. These data indicate that the mere presence of focal adhesions does not allow a reliable statement about the functional status of a cell. On the other hand, when focal adhesions are affected it is an excellent monitor of disturbed cell function.