HYPERACTIVE AUTOCRINE LOOP MEDIATED BY A NDF-RELATED FACTOR IN NEOPLASTIC HAMSTER-EMBRYO FIBROBLASTS EXPRESSING AN ACTIVATED CPH ONCOGENE

We described previously the characterization of a novel oncogene, cph, activated in primary Syrian hamster embryo fibroblasts by exposure to 3-methylcholanthrene (Velasco et al., Oncogene 9: 2065-2069, 1994). T he present report describes the participation in the neoplastic conver sion of cph-expressing (81C=39) hamster fibroblasts of a hyperactive a utocrine loop involving a nea differentiation factor [NDF]-like protei n, The tyrosine phosphorylation of the p185(erbB-2) receptor in the hu man breast carcinoma MDA-MB-453 cells was stimulated by conditioned me dium from neoplastic 81C39 cells. The extent of this stimulatory effec t was much greater than that induced by conditioned medium from normal 84-3 hamster cells. The p185(erbB-2) tyrosine phosphorylation-stimula ting activity was partially blocked by the heparin analogue pentosan p olysulfate [PPS], a known antagonist of p185(erbB-2) ligands, and was partially purified from 81C39 conditioned medium by heparin-Sepharose chromatography. The level of p185(erbB-2) tyrosine phosphorylation-sti mulating activity in the heparin-Sepharose fractions correlated direct ly with their content in NDF-like protein as immunodetected with an an ti-rat NDF antibody, Consistently, the steady-state level of NDF-relat ed mRNA was found to be four times greater in neoplastic 81C39 cells t han in normal 84-3 cells. However, the levels of erbB-2 mRNA were simi lar in both cell types, while the expression of erbB-4 mRNA was upregu lated in the neoplastic fibroblasts. The ability of 81C39 conditioned medium to stimulate protein tyrosine phosphorylation and to induce oth er PPS-sensitive growth responses on 81C39 cells themselves suggested the involvement of an autocrine loop in their neoplastic conversion, T he participation of a NDF-related factor in this autocrine loop was co nfirmed by the ability of an anti-NDF antibody to block the mitogenic activity present in their own conditioned medium. The involvement of t he cpk oncogene in the upregulation of NDF-related expression was evid enced when cph-transformed NIH3T3 fibroblasts showed elevated levels o f NDF-related mRNA, and their conditioned medium induced tyrosine phos phorylation on MDA-MB-453 cells, reproducing the effect of the medium from 81C39 hamster cells.