Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action and provide the signalling basis for mesenchyme-epithelial cross-talk. Two locally expressed factors, pleiotrophin and mammary-derived growth inhibitor (MDGI), their hormonal regulation and proposed functions will be discussed. Pleiotrophin expression in non-tumorigenic, attachment-dependent epithelial cells leads to an attachment-independent, highly tumorigenic phenotype. The fatty acid binding protein MDGI specifically inhibits growth of normal mouse mammary epithelial cells, whereas growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth by MDGI is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobulo-alveolar structures. In parallel, MDGI stimulates its own epithelial-restricted expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression suppresses the appearance of alveolar end buds and lowers the beta-casein level in organ cultures. MDGI activity can be antagonized by epidermal growth factor (EGF); reciprocally, MDGI can suppress the mitogenic effects of EGF. An MDGI-derived C-terminal 11-amino-acid peptide is able to mimic MDGI activity in vitro. In conclusion, members of the family of fatty acid binding proteins are able to regulate mammary gland differentiation locally, and fatty acid binding is not required for this activity.

Antiprogestins possess a potent antitumor activity in hormone-dependent experimental breast cancer models. Though the underlying mechanism is not clear, induction of functional differentiation seems to be a major event. This study attempts to test directly for antiproliferative and differentiation promoting activities of antiprogestins on the normal mammary gland. To this end, whole organ cultures of mammary glands from estradiol/progesterone-primed virgin mice maintained in a serum-free medium with aldosteron, prolactin, insulin, and hydrocortisone were exposed to the antiprogestin ZK114043. A 4-day treatment of organ cultures led to a strong inhibition of epithelial DNA synthesis. In parallel, ZK114043 caused alveolar cells to acquire a more differentiated phenotype distinguished by secretory active alveoli composed of single cell layers with increased fat droplet accumulation and enhanced expression of the milk proteins beta-casein and whey acidic protein (WAP). Particularly strong effects were found on the expression of mammary-derived growth inhibitor (MDGI). Both half-maximal inhibition of epithelial DNA synthesis and stimulation of MDGI mRNA expression were found at about 5 ng/ml of ZK114043. Presence in the medium of 5 micrograms/ml hydrocortisone rendered antiglucocorticoid effects of ZK114043 highly unlikely. Furthermore, prevention of action of ZK114043 by the progesterone agonist R5020 and ZK114043 stimulated expression of beta-casein and MDGI mRNA in cultured glands of 10-week-old unprimed virgin mice suggest a progesterone receptor-mediated mechanism of antiprogestin action. Two other antiprogestins, Mifepristone and Onapristone, likewise stimulated MDGI expression. The data provide direct evidence that antiprogestins act like a differentiation factor in the normal mammary gland.

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGF alpha showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGF alpha also play a role in mammary gland physiology during pregnancy by final developing and maintenance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA2-arachidonic acid signalling cascade.

Expression of proliferating cell nuclear antigen (PCNA) and c-erbB-2 oncoprotein has been assessed in 471 women with breast cancer to evaluate their prognostic value as compared to conventional histopathological factors. In univariate analysis, high PCNA expression (≥20%) predicted a significantly worse survival in lymph-node-negative tumors (univariateP=0.031). However, the effect disappeared in multivariate analysis and the histological grade remained the only independent factor for this group. Despite its close correlation to histological grade (P<0.001), PCNA expression discriminated subsets with different survival within the heterogeneous group of moderately differentiated tumors (univariateP=0.073, multivariateP=0.075). PCNA expression was not found to be a significant prognostic factor in lymph-node-positive tumors, thus it was of limited value for breast cancer patients as a whole. c-erbB-2 protein overexpression was associated with a worse survival (univariateP=0.019, multivariateP=0.057) for the entire group of patients. The effect was mainly attributed to the significance of c-erbB-2 as an independent factor in lymph-node-positive (up to three nodes, multivariateP=0.04; four or more nodes: multivariateP=0.017) and large tumors (>2 cm: multivariateP=0.002). c-erbB-2 was without significance in lymph-node-negative patients. Though both factors might amplify the prognostic information for distinct patient subsets they do not achieve the strong prognostic value of conventional histopathological features in breast cancer.

From a mouse genomic library we isolated and characterized a gene, Fabph1, encoding mammary-derived growth inhibitor (MDGI)/heart fatty-acid-binding protein (H-FABP). Exon sequences were identical with a MDGI-encoding cDNA isolated previously from the mammary gland of pregnant mice. The product of this gene has also been detected in heart, where it had been termed H-FABP. It has an intron/exon structure similar to other FABP-encoding genes. In addition to this expressed gene, we isolated a related intronless pseudogene, Fabph-ps, with an open reading frame which was highly conserved when compared with Fabph1. Fabph1 was positioned on chromosome (Chr) 4 using interspecies crosses. Analysis of somatic cell hybrids was used to assign Fabph-ps to Chr 10, and another MDGI/H-FABP-related sequence locus, Fabph-rs1, to Chr 8. A Mus spretus-specific related sequence, Fabph-rs2, was identified on Chr 17 by analysis of interspecies crosses. The 5′-flanking region of Fabph1 contains putative transcription factor-binding elements which could account for its constitutive expression in muscle tissue, as well as for its developmental stagedependent expression in mammary epithelium.

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.

The prognostic value of c-erbB-2 protein overexpression has been evaluated in 463 patients with operable breast cancer after a median follow-up of 66 months. Overexpression was observed in 99/463 (21%) of the breast tumors. It showed significant positive correlation to histological grade (p < 0.0001) and tumor size (p < 0.02). A relationship of borderline significance was observed between c-erbB-2 protein overexpression and negative or low estrogen receptor (ER) content. No significant correlation was found to lymph node involvement or proliferating tumor cell fraction as determined by the proliferating cell nuclear antigen (PCNA). After a median follow-up of 66 months (range 6 to 109 months), the overall survival of all patients amounted to 63%. Multivariate analysis revealed lymph node involvement, tumor size, histological grade, histological type, c-erbB-2 protein overexpression, progesterone receptor (PR) content, and oral contraceptive use as independent prognostic factors. In an univariate analysis, the overall survival amounted to 72% and 38% of tumor patients with negative and positive c-erbB-2 protein overexpression, respectively. The most significant finding is that c-erbB-2 overexpression has been recognized as an independent predictive factor in subsets of tumor patients who would be expected to have a generally poor prognosis, such as those indicating axillary lymph node involvement, large tumor size (> 2 cm), and PR negativity.

An established bovine mammary epithelial cell line (MAC-T) was used as a model to examine the effect of mammary-derived growth inhibitor on mammary epithelial cell proliferation. Prior to each proliferation assay, cells were synchronized in G1/G0 by culturing for 24 h without serum. Flow cytometry revealed that 90% of cells were in G1/G0, 4% in G2/M, and 6% in the S-phase after serum deprivation. Approximately 2 x 10(3) cells per well were seeded onto 24-well plates. Cells were incubated for 5 to 6 d with various amounts of mammary-derived growth inhibitor (0 to 100 ng/ml). Mammary-derived growth inhibitor and medium were changed daily. Mammary-derived growth inhibitor decreased mammary epithelial cell proliferation at .1 ng/ml. Synchronization of cells in G1/G0 was necessary for inhibition of cell proliferation. Cells not arrested by serum deprivation were not responsive to mammary derived-growth inhibitor. Inhibition of cell proliferation was transient and observed up to 96 h in culture. Mammary-derived growth inhibitor appears to act in vitro by inhibiting the resumption of stationary cell proliferation following starvation.

A method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2-3 d, and the others at 4-8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2-3 or 4-8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.

The prognostic significance of the epidermal growth factor receptor status (EGF-R-status) for certain human tumors requires the development of antibodies useful for clinical application. We used purified receptor preparations to generate monoclonal antibodies immunoreactive with the EGF-R purified from placenta membranes and A431 tumors. Four of the hybridomas contained antibodies (R2, R3, R5, and R9) which recognized both antigens. Antibody R3 was shown to display the following properties: it binds with a K(D) value of about 10(-9)-10(-10) M to the receptor, a half maximal inhibition of EGF-binding is achieved at 5 x 10(-8) M, and in Western blots of cell membranes R3 specifically detects the EGF-R at 0.1-mu-g/ml. R3 inhibits EGF-dependent clonogenic growth of NRK cells and completely blocks EGF stimulated autophosphorylation of the receptor. Moreover, R3 also detects EGF-R in paraffin-embedded tissue sections taken from human salivary gland, term placenta, and adult skin and mammary carcinomas. Thus, R3 can be used in retrospective diagnostic clinical studies and might help to develop new immunotherapeutic intervention.

Production of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating-pregnant and nonlactating-pregnant (13–44 d after cessation of milking) Holstein cows was examined. High levels of MDGI were detected in mammary tissue obtained from cows which were concurrently lactating and pregnant. A reduction in MDGI production was observed in nonlactating-pregnant cows. Key words: Bovine, MDGI, lactation, dry period, pregnancy

Expression of mammary-derived growth inhibitor in tissue from lactating and involuting bovine mammary glands was investigated. Seventeen lactating, pregnant (220 to 272 d in gestation) cows were divided in two groups of 8 and 9 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group (9 involuting cows) were slaughtered at 13 to 52 d following sudden cessation of milking. High concentrations of mammary-derived growth inhibitor (.63% of the total protein) were detected in mammary tissue of lactating cows. Mammary-derived growth inhibitor (< .10% of the total protein) was dramatically reduced during most of the involution period (13 to 45 d following cessation of milking). Mammary-derived growth inhibitor was again detected (.28% of the total protein) during the last stage of the involution (46 to 53 d after cessation of milking), which coincided with colostrum formation. When steady state concentrations of mammary-derived growth inhibitor mRNA were examined, the results obtained mirrored those obtained at the protein concentration. These data suggest that regulation of mammary-derived growth inhibitor occurs via modulation of the steady state concentration of its mRNA. Furthermore, there is a strong correlation between mammary-derived growth inhibitor expression and lactation in dairy cows.

The natural tyrosine kinase inhibitor erbstatin and a synthetic analog (1302) were co0pared for their inhibitory activity on EGF receptor kinase, PDGF receptor kinase and a src-type kinase from bovine brain. Erbstatin inhibited both growth factor receptor kinases equally well and had little effect on the src kinase. The analog exhibited similar potency for inhibition of purified EGF receptor tyrosine kinase as erbstatin, however, was clearly less effective for inhibition of purified PDGF receptor kinase as well as the src-type kinase. The selectivity was, however, not seen when the derivative was assayed with respect to inhibition of autophosphorylation of both growth factor receptors in Swiss 3T3 cell membranes. The latter finding might explain a lack of selectivity of the compound for inhibition of DNA synthesis in 3T3 cells when the cells were stimulated comparatively with EGF, insulin, EGF plus insulin or PDGF. The results suggest that the environment of growth factor receptors in the cell membrane can remarkably modify their susceptibility to tyrosine kinase inhibitors.

The clonogenic activity of a previously purified 43 kDa EGF-related protein (16) was estimated in the urine of breast cancer patients. Increase of activity was statistically significant in lymph node-positive patients, in a group of patients with larger carcinomas, with accelerated tumor growth, in premenopausal patients and younger age and in estrogen receptor negative patients. In 31 patients the activity was estimated during polychemotherapy before surgery. Differences between the values at the begin and the end of treatment were compared between all groups by the Kruskal-Wallis test (p = 0.02). Patients with progressive disease showed increasing activities (mean values from 315 to 811) while in those with complete remission activity decreased (from 449 to 213). Partial- and non responders showed no change. In a long-term follow-up study with 25 patients the pre- and postoperative activities were estimated. In 17 of 18 patients who had no local recurrence or metastasis the activity declined after surgery, whereas in 6 of 7 patients who died, activity increased 2-5 fold prior to death. A life table analysis with a total of 101 patients revealed a trend to shorter survival in the group with higher activity (p = 0.042). These observations suggest a role for the EGF-like growth factor activity in the expression of the malignant phenotype and may have significance for breast cancer diagnosis and prognosis.