Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7.

Abstract

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is, a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 560C but unstable at 1OOC.The apparent molecular weights of AR and N-Glycanasetreated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and 22,500 and %14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is =7.8. The aminoterminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.Cellular growth and differentiation appear to be initiated, promoted, maintained, and regulated by a multiplicity of stimulatory, inhibitory, and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism is a fundamental cause of growthrelated diseases including neoplasia (1-11). Physiological regulators of cell growth and differentiation, such as peptide growth factors (2, 4, 10, 11), hematopoietic regulatory proteins (5, 12-14), tumor necrosis factor types a and ,3 (8, 9), interferons (15) . In addition, PMA also alters the morphology of MCF-7 cells and PMA-treated cells exhibit the morphological characteristics of secretory cells (27,28 (vol/vol) heat-inactivated fetal bovine serum, L-glutamine, penicillin (60.6 Jug/ml), and streptomycin (100 ,ug/ml). Growth Modulatory Assay with ',I-Labeled DeoxyuridineIncorporation into DNA. The assays were performed in 96-well flat-bottomed plates (Falcon, catalog number 3072). Human epidermoid carcinoma of the vulva cells (A431) were used as test cells for growth inhibitory activity (GIA) and human forearm fibroblasts (SS) were used as indicator cells for growth stimulatory activity. A total of 3.5 x 104 cells in 50 tkl of DMEM, supplemented with 5% (vol/vol) heatinactivated fetal bovine serum, penicillin (60.6 pug/ml), streptomycin (100 tug/ml), and glutamine (test medium), was placed in all wells except peripheral wells. Three hours later, 50 jal of the test sample in test medium was added to each well; control wells received only 50 ,il of test medium. Three wells were used for each concentration of test sample. Plates were incubated at 370C for 2-3 days. After this, 100 t1d of solution of 125I-labeled deoxyuridine (Amersham) [4 Ci/mg; 0.5 Ci/ml (2 ,ul/ml in test medium); 1 Ci = 37 GBq] was added to each well and plates were incubated at 370C. After 4-6 hr, samples were processed as...

Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

“…Whereas AR was shown to induce an important stimulation of normal cell proliferation, the levels of both uPA and PAi-1 secreted by these cells were affected neither by AR, nor by EGF. On the contrary, AR induced a dose-dependent accumulation of uPA and PAi-1 in conditioned culture medium of MCF-7 and MDA-MB231 cells but failed, as previously reported by Shoyab et al (1988), to regulate their proliferation. It has to be noted that EGF was less effective than AR in stimulating protease production by tumoral cells.…”

“…Alternatively, amphiregulin peptide quantities produced by tumour cells might be influenced by transcriptional or post-transcriptional events such as impaired stability of AR mRNA, and/or by the concomitant accumulation in conditioned medium of proteases that degrade AR. Moreover, various isoforms of AR that differ in the degree of glycosylation and NH 2 -terminal processing have been described (Shoyab et al, 1988;Johnson et al, 1993b;Martinez-Lacaci et al, 1995). Therefore, the production by tumoral cells of particular AR isoforms undetectable by our enzyme-immunoassay, cannot be excluded and is presently under investigation.…”

“…Amphiregulin is a heparin-binding growth factor initially purified from conditioned medium of breast cancer epithelial MCF-7 cells treated with phorbol 12-myristate 13-acetate (Shoyab et al, 1988). Whereas AR binds and activates EGF-receptor (c-erb B1), direct interaction of AR with other members of the c-erb B family has not been reported (Johnson et al, 1993a).…”

T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells.

“…Amphiregulin, a heparin-binding growth factor structurally related to HB-EGF (6, 13), was not a SMC mitogen (data not shown) and was a relatively poor mitogen for 3T3 cells (data not shown; ref. 13) and therefore could not be responsible for the peak 1 mitogenic activity shown in Fig. 1 B and C. An anti-HB-EGF neutralizing antibody which inhibited purified U-937 cell-derived HB-EGF growth factor activity for 3T3 cells by >80% also neutralized peak 1 mitogenic activity to the same degree (data not shown).…”

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

“…Amphiregulin is a heparin-binding glycoprotein that has originally been isolated from conditioned media of human breast carcinoma MCF-7 cells and identified as epidermal growth factor receptor ligand (Shoyab et al, 1988). During development, amphiregulin is the most abundant epidermal growth factor-like growth factor in the pubertal mammary gland and has an important role in ductal morphogenesis (Luetteke et al, 1999).…”