Mammary-derived Growth Factor Research Articles

Involvement of growth factors in the regulation of pubertal mammary growth in cattle.

Pubertal mammary growth in heifers is dependent on interactions of many hormones and growth factors of which some are stimulatory while others are inhibitory. Although estrogen and growth hormone (GH) are of primary importance, more recent studies have suggested a role for both systemic and mammary tissue-specific growth factors. Growth factors may act as mediators of estrogen and GH or through specific effects of their own. These growth factors include insulin (INS), IGFs (IGF-I and IGF-II), epidermal growth factor (EGF), FGFs (FGF-1 and FGF-2), TGFs (TGF-alpha and TGF-beta's, amphiregulin (AR), platelets derived growth factor (PDGF), and mammary derived growth factor-1 (MDGF-1). Using mammary epithelial cells derived from prepubertal heifers and cultured in three-dimensional collagen gels as an in vitro model, we have investigated the mitogenic effects of a number of different growth factors (IGF-I, des(1-3) IGF-I, IGF-II, INS, EGF, TGF-alpha, AR, FGF-1, FGF-2, and TGF-beta 1). As expected, IGF-I, des(1-3)IGF-I, IGF-II and INS all stimulated proliferation of mammary cells with des(1-3)IGF-I being the most potent and INS the least potent. The mitogenic effect of IGF-I could be inhibited by both IGFBP-2 and IGFBP-3 showing that these binding proteins modulate the bioactivity of IGF-I in the mammary gland at the cellular level. Regulation of IGF availability by IGFBPs in the extracellular environment therefore is critical for IGF action in the mammary gland. Proliferation of mammary epithelial cells was also stimulated by growth factors of the EGF family, i.e. EGF, TGF-alpha and AR, however, not as much as growth factors from the IGF family. Members of the fibroblast growth factor family showed various mitogenic activities. FGF-1 stimulated DNA synthesis while FGF-2 in concentrations above 10 ng/ml inhibited DNA synthesis. TGF-beta 1 at very low concentrations stimulated proliferation slightly whereas higher concentrations strongly inhibited proliferation of mammary epithelial cells and inhibited mitogenesis induced by growth factors of both the EGF- and IGF family. This shows that TGF-beta 1 is a very potent regulator of pubertal mammary growth.

THE BIOLOGY OF BREAST CANCER

This article focuses on the major hormones and growth factors for which a critical role in normal mammary growth has been clearly defined. Certainly other hormonal systems and growth factors could also affect breast cancer initiation and progression, but their exact contribution to normal and/or malignant breast cell growth is poorly delineated. Examples of such factors include somatostatin, mammostatin, mammary-derived growth inhibitor (MDGI), mammary-derived growth factor-1 (MDGF-1), inhibins, activins, androgens, glucocorticoids, vitamin D, thyroid hormones, ecosinoids, and oxytocin. Clearly, the hormonal regulation of breast cancer cell growth and survival is multifaceted and very complex. In particular, the effects of estrogens and anti-estrogens on breast cells may depend on their interaction with a wide variety of other pathways. In addition, these interactions may vary among individual breast tumors depending on other genetic changes in the tumor cells that have not been discussed here, such as oncogene activation and loss of tumor suppressors. A more detailed understanding of how cells circumvent a dependency on these pathways is greatly needed in order to identify new biological targets and to design novel therapies for breast cancers that are resistant to anti-estrogen therapy. Such agents could be used alone or in combination with anti-estrogens to improve response to a second course of hormonal therapy.

Production and localization of mammary-derived growth factor 1 in the normal and malignant mammary gland

Previously, we have detected a human milk and breast cancer-derived, acidic, 62 kDa, N-glycosylated growth factor named mammary-derived growth factor 1 (MDGF1). The N-terminal amino acid sequence of MDGF1 showed it to be a new growth factor. The factor stimulated collagen synthesis by fibroblasts and proliferation in normal and malignant mammary epithelial cells. Polyclonal antisera raised against the N-terminal synthetic peptide has been used to demonstrate that some human breast cancer and normal mammary epithelial cell strains and cell lines secrete the factor into their conditioned media. The antibody could adsorb the biological activity of the factor present in conditioned media. The objective of the present study was to utilize this high titre polyclonal anti-MDGF1 antisera to establish the presence and distribution of the factor in benign and malignant human breast tissues as determined by immunohistochemical analysis and enzymelinked immunosorbent assay (ELISA) assays. Frozen sections of normal and malignant breast tissue were used for immunostaining, while homogenized soluble fractions were used for ELISA testing. Antigen absorption confirmed the antibody staining to be specific. MDGF1 is localized in the duct and lobular epithelial cells of the mammary gland. Staining was nuclear and cytoplasmic in tumour and normal specimens. All of the breast tumour specimens demonstrated intense staining of the tumour cells. Localization of MDGF1 in benign and malignant human breast tissue suggests that it is universally present in breast epithelial derived cells. Study of MDGF1 could eventually provide new insights into breast development and/or tumour formation and progression.

Receptor-induced phosphorylation by mammary-derived growth factor 1 in mammary epithelial cell lines.

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.

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines

A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell-surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth-promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell-conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of mammary-derived growth factor 1 receptors and response in human mammary epithelial cell lines.

Mammary-derived growth factor 1 (MDGF1, with a molecular mass of 62 kDa and pI of 4.8) has been purified from human milk (Bano, M., Salomon, D. S., and Kidwell, W. R. (1985) J. Biol. Chem. 260, 5745-5752). N-terminal sequence of 18 amino acids showed no homology to any known growth promoting peptides. The present study was carried out to evaluate the biological effects of and binding sites for MDGF1 on normal human and breast cancer cell lines. At a concentration of 10-25 ng/ml the factor stimulated the growth of estrogen receptor-positive MCF-7 human breast cancer cells by 50% and also stimulated the synthesis of collagen IV by 40%. It did not have any effect on ZR75-1, T47-D, and MDA-MB 231 cells. The factor showed a biphasic effect on the estrogen receptor-negative MDA-MB 468 cells at concentrations above 5 ng/ml. The growth of normal human mammary epithelial cells (184 strain) was enhanced by 35%, whereas immortalized non-tumorigenic 184A1N4 human mammary epithelial cells were stimulated by about 60-70%. However, transformation of these cells by SV40-T, v-Ha-ras, or v-mos desensitized them to MDGF1. Iodinated MDGF1 binds to moderate affinity sites on the responsive MCF-7, MDA-MB 468, and 184A1N4 cell lines (KD = 6 x 10(-9) M). Cross-linking of 125I-MDGF1 to binding sites revealed the presence of a major band of molecular mass of approximately 180-200 kDa in MCF-7 and MDA-MB 468 cell lines. Labeling of this band was inhibited by excess unlabeled MDGF1. These data suggest that human mammary epithelial cell lines possess receptors for MDGF1.

Local effects of EGF, alpha-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in vivo.

Five-week-old female mice supplemented with estradiol and progesterone are able to respond to epidermal growth factor (EGF) and EGF-like growth factors (alpha-transforming growth factor [alpha-TGF] and crude mammary-derived growth factor) with local lobuloalveolar development when these growth factors are directly introduced into the mammary glands via slow-release cholesterol-based pellets. Contralateral glands receiving pellets containing only cholesterol showed no growth response. The local growth effect is maximal at 4-5 days of exposure to hormones and growth factors. The glands appear to be more sensitive to alpha-TGF than EGF, since local development is seen with one-fifth the level of the former vs. the latter growth factor and can be seen even in the absence of the systemic estrogen/progesterone supplement.

Purification of a mammary-derived growth factor from human milk and human mammary tumors.

A growth factor, mammary-derived growth factor 1 (MDGF1), has been purified to apparent homogeneity from human milk. The factor is a pepsin-sensitive, reducing agent-insensitive protein with a molecular mass of 62 kDa and a pI of 4.8. An apparently identical factor has been isolated from human mammary tumors, suggesting that MDGF1 might be made by and act as an autocrine growth factor for mammary cells. High affinity receptors for MDGF1 have been detected on mouse mammary cells, normal rat kidney cells, and A431 epidermoid cells (KD = 2 X 10(-10) M). MDGF1 at picomolar levels stimulates the growth of mammary cells and greatly amplifies their production of collagen, apparently via elevating collagen mRNA levels, an effect that is demonstrated for normal rat kidney cells. The responsiveness of mammary cells to MDGF1 is attenuated when the cells are grown on a basement membrane collagen substratum, a component of the extracellular matrix upon which these cells normally rest in vivo. MDGF1 thus may regulate the production of new basement membrane as mammary epithelium invades the stroma during proliferation.