Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity.

Abstract

Treatment of sparse, proliferating cultures of [Swiss mouse fibroblast] 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using 3 assay systems: assessment of total cell number; measurement of [3H]thymidine incorporated into acid-precipitable DNA; and determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no difference in the average number of trains per labeled cell in the two cultures. The inhibitory effect of the CM on target cell proliferation was reversible. This growth inhibitory activity was collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. A system was developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.