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Abstract
Removal of chicken embryo fibroblasts infected with the temperature-sensitive mutant of Rous sarcoma virus (RSV-CEF) from their substrata with the calcium chelator, EGTA, leaves behind the substratum associated material (SAM or extracellular matrix). We have studied the proteins/glycoproteins and the glycosaminoglycan, hyaluronic acid (HA), in SAM during the development of the transformed state and have: (1) detected a novel, low molecular weight protein (M(,r) (TURNEQ) 21,000) in SAM; (2) biochemically analyzed this 21K protein; (3) characterized transformation-sensitive alterations in HA; and (4) investigated the role of HA in modifying cell-substratum adhesion.
Increased synthesis and deposition of the 21K protein into SAM is oberved within hours of transfer of RSV-CEF to the permissive (transforming) temperature. Characterization reveals metabolic labeling by amino acids, but not mannose or phosphorous. It is resistant to extraction by EGTA, urea and several detergents except sodium dodecyl sulfate. The 21K protein is sensitive to Pronase and chymotrypsin but insensitive to trypsin or collagenase. It is resistant to removal from the substratum by cell-associated proteolytic or glycolytic enzymes. The 21K protein appears to be shed from the cell surface, possibly as part of a glycoprotein/glycosaminoglycan complex and then becomes strongly associated with the substratum.
Elevated amounts of hyaluronic acid are synthesized during the early stages of transformation. We have detected several alterations in both amounts and organization of HA during this process. The greatest alteration is in a high molecular weight species, which is 2-fold higher in SAM and approximately 20-fold higher in a "loosely-associated" HA-species. Cell surface distribution of HA is also altered upon transformation. In nontransformed RSV-CEF, 75% of the cell surface HA is in SAM. Upon transformation this falls to 62% as a result of increased "loosely-associated" HA.
Alterations in cell surface HA upon transformation coincide with the loss of cell-substratum adhesion. Treatment of transforming cells with hyaluronidase (specifically degrades HA) results in stronger cell-substratum adhesion. Exogenously added HA only slightly affects initial attachment of cells to the substratum. HA does not appear to affect cell morphology. Results suggest that HA may be involved in retarding normal cell spreading and promoting the ability of transformed cells to detach.
