Abstract
Previous work has shown that a mammary-derived growth factor (MDGF1), a human milk-derived, acidic, 62-kDa, N-glycosylated growth factor binds to cell surface receptors and stimulates proliferation of mammary epithelial cells. An 18-amino acid N-terminal partial sequence of the factor did not show any homology to other known growth factors or proteins. Using polyclonal antiserum raised against the synthetic peptide, we demonstrated that conditioned medium prepared from human breast cancer cell lines contains the factor. The antibody could adsorb the biological activity of the factor present in the conditioned medium. Earlier experiments on receptor cross-linking indicated that the receptor was approximately 120-140 kDa. Since tyrosine phosphorylation plays a crucial role in cell proliferation and cell transformation, experiments were conducted to find out whether MDGF1 induces the appearance of phosphotyrosine in MDGF1-receptor-positive MDA-MB 468, MCF-7, and 184A1N4 cell lines compared to receptor-negative lines. Western blot analysis using monoclonal antiphosphotyrosine indicated that MDGF1 induces phosphotyrosine in a 180-185-kDa protein in MDGF1 receptor-positive cell lines. Phosphorylation was not blocked and phosphorylated proteins were not immunoprecipitated by an antibody directed against the binding site of the EGF receptor. Cell membrane fractionation demonstrated that phosphorylation induced by MDGF1 was membrane-associated. The nature of this 180-185-kDa protein and its possible relationship to the MDGF1 receptor are under investigation.
Highlights
Previous work hasshown that a mammary-derived size, PI, amino acid content, primary protein sequence, and growth factor (MDGFla),human milk-derived, acidic, disulfide bond reducing agent sensitivity
Using polyclonal antiserum raised against the synthetipceptide, we demonstrated that conditioned medium prepared from human breast cancercell lines containsthe factor
Western blot analysis using monoclonal antiphosphotyrosine indicated that MDGF1induces phosphotyrosinein a 180-186-kDaproteinin MDGFl receptor-positive cell lines
Summary
MDGFl induces the appearanceof phosphotyrosine in cells and increased their production of collagen. The receptors for EGF, platelet-derived growth factor, insulin, and insulinlike growth factor 1, among others, are known to possess tyrosine kinase activity. The normal 184cell strainand 184AlN4 (a carcinogen-immortalized subclone) were obtained from Dr Martha Stampfer (Berkeley, CA) and maintained inimproved minimal essential medium in 0.5%fetal calf serum in the presence of EGF (10 ng/ ml, Collaborative Research), insulin (10 pg/ml, Sigma, St. Louis, MO), transferrin (10 pg/ml, Sigma), and hydrocortisone (0.5 pg/ml, Sigma) according to thepublished protocol [15]. Filters were preincubated with 3% bovine serum albumin in Tris-buffered saline containing Tween (TBST, 10 mM Tris-HC1, pH 7.5, 150 mM NaC1,0.05% Tween 20) overnight a t 4 “C.Blots were incubated with anti-phosphotyrosine (2 pg/ml) for 3 h a t room temperature.
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