Growth factor-independent proliferation of rat mammary carcinoma cells by autocrine secretion of neu-differentiation factor/heregulin and transforming growth factor-alpha.
Serially transplantable rat mammary tumor (RMT) cells are not dependent on exogenous epidermal growth factor (EGF) and insulin-like growth factor-I for continuous growth in serum-free medium. Previously, we found that conditioned medium obtained from these cells contained EGF-like mitogenic activity and stimulated tyrosine phosphorylation of a 185-kDa protein in EGF-dependent mammary epithelial cells. This protein is distinct from the EGF receptor and resembles a 185-kDa tyrosine-phosphorylated protein present in RMT cells themselves. The results of the studies reported here indicate that the tyrosine-phosphorylated p185 detected in growth factor-independent RMT cells and in human mammary epithelial cells exposed to RMT-conditioned medium was activated erbB-2 protein. Partial purification of the activating factor present in RMT-conditioned medium yielded a heparin-binding growth factor with biochemical properties similar to those of neu differentiation factor/heregulin (NDF/HRG). RNA-polymerase chain reaction analysis demonstrated that RMT cells expressed mRNA for NDF/HRG, and western-blot analysis confirmed the presence of the 45-kDa secreted form of NDF/HRG in conditioned medium from the growth factor-independent RMT cells. The biological activity of partially purified rat NDF/HRG was examined and found to be the same as that of the pure growth factor. In addition, we found that RMT-conditioned medium, fractionated on an anion-exchange column and by reverse-phase high-pressure liquid chromatography, contained a potent EGF-like growth factor that was distinct from NDF/HRG. This factor competes with 125I-EGF for binding to EGF receptors and has an apparent molecular mass of 6600 Da. This factor copurifies by high-pressure liquid chromatography with pure transforming growth factor-alpha (TGF-alpha), and the cells are positive for TGF-alpha mRNA. Thus, growth factor-independent RMT cells also synthesize and secrete TGF-alpha. These results indicate that growth factor-independent cells secrete two growth factors with overlapping biological activities and suggest that autocrine loops mediated by these factors are important in the growth factor-independent proliferation of the RMT cells.
Ethier
S P
SP
Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical School, Ann Arbor, USA.
Langton
B C
BC
Dilts
C A
CA
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001940:Breast; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000096:biosynthesis / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000473:pathology*; D008969:Molecular Sequence Data; D020880:Neuregulins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D016133:Polymerase Chain Reaction; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D017931:DNA Primers; D004815:Epidermal Growth Factor; D006023:Glycoproteins; D006133:Growth Substances; D020880:Neuregulins; D019000:Phosphotyrosine; D018719:Erb-b2 Receptor Tyrosine Kinases; D016211:Transforming Growth Factor alpha