Secretion of neu differentiation factor-like polypeptides by cph-transformed fibroblasts: cloning and characterization of Syrian hamster neuregulin cDNAs.

We reported previously that expression of the cph oncogene in Syrian hamster and mouse cells leads to the secretion of a polypeptide factor or factors structurally and functionally related to neu differentiation factor (NDF) and the establishment of an autocrine loop mediated through the simultaneous overexpression of the erbB4 receptor. To identify the nature of this factor and to characterize its role in the neoplastic conversion of Syrian hamster embryo cells, we cloned cDNAs hybridizing to rat NDF-derived probes by screening a library prepared from neoplastic 81C39 cells, which harbor an activated cph oncogene and secrete active NDF-like polypeptides. Sequence analysis of the isolated clones revealed a high level of homology between the hamster neuregulin and its rat and human counterparts and the existence of various neuregulin cDNA variants in Syrian hamster cells, presumably originating from alternatively spliced RNA species. Interestingly, some of these neuregulin cDNAs were longer (up to 5.5 kb) than those isolated before from any other system, suggesting that the Syrian hamster neuregulin precursor mRNA accumulates at greater concentrations than in other species. We also detected different hamster neuregulin protein isoforms by using in vitro transcription-translation analyses. Peptide sequence analysis identified the major NDF-like polypeptide secreted by 81C39 cells as an alpha2b-neuregulin. Northern blot analyses with the cloned cDNA inserts showed that neuregulin overexpression was commonly associated with the neoplastic conversion of chemically initiated hamster embryo fibroblasts. This, along with the detection of elevated erbB2- or erbB4-specific transcripts in most (six of eight) neoplastic cell lines tested, supports the notion that autocrine neuregulin signaling plays an important role in maintaining the transformed phenotype by providing a growth advantage to cph-transformed cells.