Transferrin is an autocrine growth factor secreted by Reuber H-35 cells in serum-free culture

We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [35S]methionine. A 1:4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59 +/- 0.12 X 10(5) to 3.35 +/- 0.34 X 10(5) after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P less than 0.05) less growth (2.14 +/- 0.41 X 10(5) cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [35S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1 +/- 1.2 ng of transferrin was secreted per 10(6) cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P less than 0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin.