Loss of different pericellular matrix components of rat cells transformed with a T-Class ts mutant of rous sarcoma virus

Abstract

Rat cells transformed by the T-class ts mutant LA339 derived from RSV B77 (2R cells) and the parental nontransformed Rat-1 cells were analyzed for the production and deposition of pericellular matrix components as a function of growth temperature. The cells were incubated at the permissive (35°) and nonpermissive (39.5°) temperatures for transformation and the collagenous and noncollagenous (fibronectin and laminin) matrix proteins were studied by polypeptide analysis of the metabolically labeled cells and their culture media. Immunofluorescence analysis of the pericellular matrix structures was performed using specific antibodies against laminin, fibronectin, and heparan sulfate proteoglycan. At 39.5° dense pericellular matrices were detected in both 211 and Rat-1 cell layers, whereas at 35° the 2R cells lost their pericellular matrix structures. The cells continued to synthesize and secrete fibronectin, laminin, and heparan sulfate proteoglycan as well as slightly reduced amounts of procollagen into the culture medium but failed to deposit them in a matrix form. Another major temperature-sensitive difference in the secreted polypeptides was the production by the 211 cells at 35° of a 56,000-dalton protein, similar to that reported in other transformed cell cultures. The present findings indicate that the transformed rat 2R cells lose, upon activity of the pp60 src, their capacity to deposit the synthesized matrix components into pericellular structures.