• autocrine growth, EGF independence


To determine whether the enhanced expression of transforming growth factor α (TGFα) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGFα cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGFα mRNA expression by northern blot analysis, TGFα secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGFα-transfected NOG-8 clones expressed high levels of a specific TGFα mRNA, secreted elevated levels of TGFα into the culture medium (177–595 ng/108 cells/48h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGFα peptide was able to inhibit colony formation in soft agar by TGFα-transfected NOG-8 clones that produced high levels by TGFα This inhibition suggested that TGFα acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGFα, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGFα cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.