No abstract

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrinmediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activatedsrc over-expression system. In the present study, we describe the identi®cation of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src speci®c tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identi®ed as bona-®de src substrates, cortactin and Sam68. UCS15A diered from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to dier from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory eects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway. Oncogene (2001Oncogene ( ) 20, 2068Oncogene ( ± 2079

No abstract

No abstract