Transferrin Is an Autocrine Growth Factor Secreted by Reuber H-35 Cells in Serum-Free Culture

• Published in: In Vitro Cellular & Developmental Biology 1989-07, Vol.25 (7), p.650-654
• Main Authors: Shapiro, Lawrence E., Wagner, Neil
• Format: Article
• Language: English
• Subjects: Animal cells; Animals; Antibodies; Biological and medical sciences; Biotechnology; Cell culture techniques; Cell cultures. Hybridization. Fusion; Cell Division; Cell growth; Cell lines; Cultured cells; Dialysis; DNA - biosynthesis; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Fundamental and applied biological sciences. Psychology; Hepatocellular carcinoma; Hepatocytes; Immunoglobulin G; Immunoprecipitation; Immunosorbent Techniques; Liver Neoplasms, Experimental - pathology; Liver Neoplasms, Experimental - secretion; Methods. Procedures. Technologies; Molecular and cellular biology; Radioimmunoassay; Transferrin - immunology; Transferrin - pharmacology; Transferrin - secretion; Transferrins; Trypsin; Tumor Cells, Cultured
• ISSN: 0883-8364; 0073-5655; 2327-431X; 1475-2689
• DOI: 10.1007/BF02623636

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 × 10⁵ to 3.35 ± 0.34 × 10⁵ after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P < 0.05) less growth (2.14 ± 0.41 × 10⁵ 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 10⁶ cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P < 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.