Human A673 cells secrete high molecular weight EGF-receptor binding growth factors that appear to be immunologically unrelated to EGF or TGF-?

Abstract

Extracts of serum-free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio-Gel P-100 and carboxymethyl (CM)-cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reversephase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage-independent growth (AIG) activity that coeluted with 25–26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM-C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21–22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF-α- (hTGF-α), or rat TGF-α (rTGF-α) and failed to give positive signals in Western blots under conditions in which TGF-α was readily detected when using an antisera raised against the 17 C-terminal amino acids of rTGF-α. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to M_r 15,000 and 22,000 in the 25–26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21–22% acetonitrile eluate. The presence of multiple forms of EGF-receptor-binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF-α family of growth-promoting polypeptides.