No abstract

Summary The interferons are a group of naturally occuring proteins that inhibit the growth of tumours in vivo and many transformed cell lines in vitro. The mechanisms of action of interferon, however, remain unclear. The IFN induced inhibition of growth of many epithelial cancer cell lines is associated with changes Interferons are a family of secretory cellular proteins with a wide range of biological effects. In addition to their antiviral activity, IFNs inhibit growth of both normal and transformed cells (Balkwill et al., 1982; Bradley & Ruscetti, 1981 (Chakravarthy et al., 1991). The purpose of the present study was to examine the effect of IFN on synthesis and stability of the EGFR protein and mRNA. Materials and methods Cell cultureThe MDA 468 cell line (kindly provided by Dr Ron Buick, Ontario Cancer Institute) was derived from a human breast carcinoma (Filmus et al., 1985). It was routinely cultured in L-15 medium (Gibco) supplemented with 10% foetal bovine serum (FBS) (Sigma, St Louis, MO). Cells were subcultured twice weekly by trypsinisation. All cells were used within 20 passages of the original stock.Protein labelling and immunoprecipitation MDA 468 cells (6 x 105 total) were plated in 75 cm2 flasks in 50% DMEM, 50% F-12 medium with 10% calf-serum. Medium were aspirated, the cells rinsed with methionine-free RPMI (ABS, Columbia, MD) and incubated for 6 h with "5Smethionine (38 piCi ml-') (S.A. 1129 Ci mmol-') (New England Nuclear, Boston, MA) in 5ml methionine free RPMI. Cells were lysed in 1 ml of 50 mM Tris HCI, Triton X-100 (1% v/v), SDS (0.1% w/v), PMSF (1 mM), EDTA (1 mM) and leupeptin (1 ,Ag ml-') at room temperature. Cell lysates were centrifuged and duplicate aliquots of each lysate were standardised by trichloroacetic acid precipitation. The samples were then incubated with a monoclonal antibody to EGF receptor (antibody 528) (final concentration of 1.5 jig ml-') (Oncogene Science Inc, Mineola, NY) or a non-specific antibody of the same isotype and Protein-A Sepharose. Bound material was released by heating the complexes in SDS buffer for 2 min at 100°C. The samples were then analysed on 7.5% SDS polyacrylamide gels. Following electrophoresis, the gels were treated with Enhance (NEN) and dried. The gels were exposed to Kodak XAR-5 film and developed. Where indicated, the relative amounts of immunoprecipitated EGF receptor were quantitated by densitometric analysis of the autoradiograms using a LKB laser Correspondence: A.W. Hamburger.

Previous studies have demonstrated that binding of peptide growth factors such as epidermal growth factor (EGF) decreases as cell density increases. We now report that binding of EGF to MDA 468 breast carcinoma cells is reduced as cells increase in density with time in culture. Cells at low density bound more EGF per cell than cells at higher density. The reduction of EGF binding was due to a reduction in receptor number. Metabolic labeling of MDA 468 cells with [³⁵S]-methionine followed by immunoprecipitation of the EGF receptor (EGFR) indicated that the amount of total receptor protein was decreased. Using RNA blot hybridization, we found that high-density cells contained decreased amounts of EGFR transcripts. Northern analysis revealed that both the 10- and 5.6-kilobase mRNA transcripts encoding the EGFR were decreased. These data suggest that increasing cell density with time in culture results in modulation of EGFR content, with changes at the level of both protein and mRNA expression.

Previous work from our laboratory has demonstrated that gamma-interferon (IFN) inhibits growth of the human breast carcinoma cell line MDA 468, while enhancing expression of epidermal growth factor receptor (EGFR). Epidermal growth factor at high levels is known to inhibit growth of this cell line. Because MDA 468 cells produce low levels of transforming growth factor (TGF)-alpha (a ligand for epidermal growth factor receptor), we reasoned that IFN-induced cytotoxicity could be partially mediated by enhanced secretion of TGF-alpha. Therefore, we determined the ability of IFN to modulate the endogenous expression of TGF-alpha by MDA 468 cells. IFN-gamma, at 500 units/ml, increased the levels of TGF-alpha in serum-free conditioned media of MDA 468 cells 3-fold as measured by radioimmunoassay. TGF-alpha mRNA was similarly increased approximately 3-fold after 5 days of IFN treatment as determined by dot blot and Northern analysis. IFN increased expression of TGF-alpha in conditioned media in a dose-dependent fashion. Increased secretion of TGF-alpha into conditioned media was not observed at Days 1 and 3. Similarly, increases in TGF-alpha mRNA were not observed at those time points. These results demonstrate that IFN-gamma enhanced secretion of TGF-alpha by MDA 468 cells. Although exogenous TGF-alpha inhibited MDA 468 cell growth, the role that the enhanced endogenous production of TGF-alpha plays in the cessation of cell growth induced by IFN remains to be determined.