Secretion of neu differentiation factor–like polypeptides bycph-transformed fibroblasts: Cloning and characterization of Syrian hamster neuregulin cDNAs

Abstract

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.

The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress

“…These studies showed that expression of the cph oncogene in Syrian hamster and mouse cells leads to the establishment of an autocrine loop mediated by the simultaneous secretion of an a2b-neuregulin and the increased expression of the erbB2 or erbB4 receptors (Avila et al, 1995;Velasco et al, 1998). These results support the notion that autocrine neuregulin signaling plays an important role in maintaining the transformed phenotype by providing a growth advantage to the cph-transformed cells.…”

“…In this regard, it is important to note that we have shown previously that one of the most immediate phenotypic consequences of the endogenous (in Syrian hamster embryo cells) or heterologous (in NIH3T3 cells) expression of the cph oncogene is the establishment of a hyperactive autocrine loop mediated by the secretion of a neuregulin and the overexpression of the erbB4 or erbB2 neuregulin receptors (Avila et al, 1995;Velasco et al, 1998). In the course of experiments designed to characterize this autocrine loop cells were maintained under conditions of serum deprivation.…”

“…In the course of previous experiments designed to characterize the hyperactive neuregulin autocrine loop operating in chemically-initiated, 81C39 neoplastic Syrian hamster embryo cells (Avila et al, 1995;Velasco et al, 1998), we maintained the cells in medium without serum for various periods of time to facilitate the puri®cation of polypeptides secreted into the culture¯uids. After serum starvation, we collected the cells, extracted RNA and performed northern hybridization analyses using cph cDNA probes.…”

Neuregulin Stimulates Myogenic Differentiation in an Autocrine Manner

“…Moreover, the inhibitory effect on primary culture was less prominent than that on L6 cell line. It may be explained by the observation that the primary cultures of rat myoblasts were significantly contaminated by fibroblasts (less than 30% of total cell number at the time of plating and more than 60% 2 days after plating), which are known to secrete NRGs (31,32). Another explanation could be the heterogeneity in the population of rat primary myoblasts, a part of which might have already committed to differentiation program at the time of the preparation of the primary cultures.…”

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N -nitrosoethylurea: high incidence but low allelic representation

“…These factors continue to act as needed during adult life for tissue renewal and wound repair. Oncogenic ras transformation of mouse ®broblasts (Alimandi et al 1995), or cph transformation of Syrian hamster embryo cells (Avila et al 1995), results in a hyperactive autocrine loop mediated by overexpression of neuregulin proteins that contribute to the transformed phenotype (Velasco et al 1998). Transformation of murine or human breast cell lines with mutation-activated or over-expressed wildtype p185 neu also results in autocrine, juxtacrine and paracrine expression of multiple neuregulin proteins (Minicione et al 1996;Ethier et al 1996;Sepp-Lorenzino et al 1996).…”