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.     

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.     

A transforming growth factor related to epidermal growth factor is expressed by fetal mouse salivary mesenchyme cells in culture

Fetal mouse salivary mesenchyme cells secrete a protein with an apparent MW of 15 Kd that is immunologically related to epidermal growth factor (EGF). Conditioned medium collected from these cells in culture stimulates the growth of primary mouse mammary epithelial cells cultured within collagen gels, competes for binding to EGF receptor sites on these mammary epithelial cells and stimulates the anchorage-independent growth of normal rat kidney fibroblast cells within soft agarose. Prior immunoprecipitation of salivary mesenchyme cell conditioned medium with anti-EGF antibodies effectively removes or attenuates all of these effects confirming that an EGF-like factor is involved in these responses.     

Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

Purification and characterization of a complex containing matriptase and a Kunitz-type serine protease inhibitor from human milk.

Matriptase, a trypsin-like serine protease with two potential regulatory modules (low density lipoprotein receptor and complement C1r/s domains), was initially purified from T-47D breast cancer cells. Given its plasma membrane localization, extracellular matrix-degrading activity, and expression by breast cancer cells, this protease may be involved in multiple aspects of breast tumor progression, including cancer invasion. In breast cancer cells, matriptase was detected mainly as an uncomplexed form; however, low levels of matriptase were detected in complexes. In striking contrast, only the complexed matriptase was detected in human milk. The complexed matriptase has now been purified. Amino acid sequences obtained from the matriptase-associated proteins reveal that they are fragments of a Kunitz-type serine protease inhibitor that was previously reported to be an inhibitor of the hepatocyte growth factor activator. In addition, matriptase and its complexes were detected in milk-derived, SV40 T-antigen-immortalized mammary luminal epithelial cell lines, but not in human foreskin fibroblasts or in HT-1080 fibrosarcoma cells. These results suggest that the milk-derived matriptase complexes are likely to be produced by the epithelial components of the lactating mammary gland in vivo and that the activity and function of matriptase may be differentially regulated by its cognate inhibitor, comparing breast cancer with the lactating mammary gland.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Identification of a new squamous cell differentiation marker and its suppression by retinoids.

Rabbit tracheobronchial epithelial cells (RbTE) can undergo squamous cell differentiation under defined culture conditions and, therefore, have been used as a model to study the regulation of squamous cell differentiation markers. In the present study, we identified a 20-kDa protein, designated rSQ20, in the serum-free growth medium conditioned by RbTE cells undergoing squamous cell differentiation. The protein was also found in extracts of squamous differentiated cells. rSQ20 was labeled by cells incubated with [35S]methionine but not with [3H]glucosamine, suggesting that it is not a glycoprotein. Undifferentiated cells did not produce this protein. rSQ20 was detected in the conditioned medium of RbTE cells after they reached a confluent and growth-arrested state, and thereafter its level increased markedly and concurrently with an increase in type I (epidermal) transglutaminase, an established marker of squamous cell differentiation. rSQ20 found in concentrated conditioned medium of squamous differentiated RbTE cells was eluted from a gel filtration column as a protein of 20 kDa, similar to that found by gel electrophoresis under denaturing conditions, suggesting that it is not a multimeric protein. A protein with an apparent molecular weight of 16 kDa (rSQ16), probably the product of partial proteolysis of rSQ20, was often found in various amounts in the conditioned medium of differentiated RbTE cells. beta-All-trans retinoic acid and other vitamin A analogues (retinoids), which suppress squamous cell differentiation, inhibited the expression of rSQ20 in RbTE cells. RbTE cells immortalized by transfection with SV40 large T antigen as well as malignantly transformed derivatives obtained from the immortalized cells by further transfection with v-Ha-ras secreted SQ20 and SQ16 when grown to high cell densities although their squamous differentiation was impaired. An analogous protein with an apparent molecular weight of 16 kDa, designated hSQ16, was detected in the medium of differentiated normal human bronchial epithelial (NHBE) cells and normal human epidermal keratinocytes (NHEK). No such protein could be detected in the medium in which undifferentiated NHBE or NHEK cells were grown. These results suggest that rSQ20 and hSQ16 are new markers of squamous cell differentiation.     

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.     

Cripto: roles in mammary cell growth, survival, differentiation and transformation

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.     

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.     

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.     

Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

Parathyroid hormone-related protein production by primary cultures of mammary epithelial cells.

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult non-neoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/10(5) cells x 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)-beta, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.     

A soluble form of Wnt-1 protein with mitogenic activity on mammary epithelial cells.

The proto-oncogene Wnt-1 plays an essential role in fetal brain development and causes hyperplasia and tumorigenesis when activated ectopically in the mouse mammary gland. When expressed in certain mammary epithelial cell lines, the gene causes morphological transformation and excess cell proliferation at confluence. Like other members of the mammalian Wnt family, Wnt-1 encodes secretory glycoproteins which have been detected in association with the extracellular matrix or cell surface but which have not previously been found in a soluble or biologically active form. We show here that conditioned medium harvested from a mammary cell line expressing Wnt-1 contains soluble Wnt-1 protein and induces mitogenesis and transformation of mammary target cells. By immunodepletion of medium containing epitope-tagged Wnt-1, we show that at least 60% of this activity is specifically dependent on Wnt-1 protein. These results provide the first demonstration that a mammalian Wnt protein can act as a diffusible extracellular signaling factor.     

Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

Human mammary cell survival following ionizing radiation

We have developed and evaluated methods of culturing defined stromal and epithelial populations of normal human breast cells. These cell populations were used to generate radiation dose/survival curves. The epithelial cell population required specific hormones, growth factors, and conditioned media, as well as fibroblast feeder layers for clonal growth. Stromal cells grew well in a less complex medium. The stromal and parenchymal cell populations of the normal human breast were characterized by light and electron microscopy, immunohistochemical human fibronectin staining, gamma glutamyltranspeptidase histochemical staining, and cell sizing. Survival curves were generated using cells from four donors. The average D0 for epithelial cells was 122 cGy, with an average n value of 2.4. The average D0 and n values for stromal cells were 114 cGy and 2.0. The survival of human breast epithelial cells is compared to that of the cells of the rat mammary gland. The D0 values of both species are essentially the same, while the n value for human epithelial cells is lower. This difference in the n value may be a species specific response to radiation, or may merely reflect a difference in the two assay systems used to generate the survival curves.     

Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells.

A full-length complementary DNA clone from a normal human mammary epithelial cell (strain 184) encoding a 25-kilodalton protein, HME1, was isolated. Expression of HME1 RNA appears to be limited to epithelial cells. The HME1 sequence has extensive sequence homology with bovine 14-3-3 protein, which is an activator of tyrosine and tryptophan hydroxylase. However, the tissue distribution, arrangement of charged amino acids, and location of potential phosphorylation sites of HME1 differ from those of 14-3-3. Compared with normal mammary epithelial cells, expression of HME1 RNA was dramatically low in two cell lines derived from human mammary carcinoma that were examined, and in a line of normal mammary epithelial cells transformed by oncogenes. HME1 therefore appears to be a cellular differentiation marker that may be down-regulated during neoplastic transformation.