ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.     

Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation.

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.     

Differential transformation of mammary epithelial cells by Wnt genes.

The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent.     

Nuclear receptors agonists exert opposing effects on the inflammation dependent survival of breast cancer stem cells

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.     

Mammary gland copper transport is stimulated by prolactin through alterations in Ctr1 and Atp7A localization

Milk copper (Cu) concentration declines and directly reflects the stage of lactation. Three Cu-specific transporters (Ctr1, Atp7A, Atp7B) have been identified in the mammary gland; however, the integrated role they play in milk Cu secretion is not understood. Whereas the regulation of milk composition by the lactogenic hormone prolactin (PRL) has been documented, the specific contribution of PRL to this process is largely unknown. Using the lactating rat as a model, we determined that the normal decline in milk Cu concentration parallels declining Cu availability to the mammary gland and is associated with decreased Atp7B protein levels. Mammary gland Cu transport was highest during early lactation and was stimulated by suckling and hyperprolactinemia, which was associated with Ctr1 and Atp7A localization at the plasma membrane. Using cultured mammary epithelial cells (HC11), we demonstrated that Ctr1 stains in association with intracellular vesicles that partially colocalize with transferrin receptor (recycling endosome marker). Atp7A was primarily colocalized with mannose 6-phosphate receptor (M6PR; late endosome marker), whereas Atp7B was partially colocalized with protein disulfide isomerase (endoplasmic reticulum marker), TGN38 (trans-Golgi network marker) and M6PR. Prolactin stimulated Cu transport as a result of increased Ctr1 and Atp7A abundance at the plasma membrane. Although the molecular mechanisms responsible for these posttranslational changes are not understood, transient changes in prolactin signaling play a role in the regulation of mammary gland Cu secretion during lactation.     

Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

ROSA26 mice carry a modifier of Min-induced mammary and intestinal tumor development

B6.129S7-Gtrosa26 (B6.R26) mice carry a LacZ-neoR insertion on Chromosome (Chr) 6, made by promoter trapping with 129 ES cells. Female C57BL/6J Apc ^Min /+ (B6Min/+) mice are highly susceptible to intestinal tumors and to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, B6.R26/+Min/+ females develop fewer mammary and intestinal tumors after ENU treatment than do B6 Min/+ mice. B6.R26/+ mice from two independently derived congenic lines show this modifier effect. Each of these congenic lines carries approximately 20 cM of 129-derived DNA flanking the insertion, raising the possibility that the resistance is due to a linked modifier locus. To further map the modifier locus, we have generated several lines of mice carrying different regions of the congenic interval. We have found that resistance to mammary and intestinal tumors in ENU-treated Min/+ mice maps to a minimum 4-cM interval that includes the ROSA26 LacZ-neoR insertion. Therefore, the resistance to tumor development is due to either the ROSA26 insertion or a very tightly linked modifier locus. Received: 10 May 2000 / Accepted: 25 July 2000     

Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody. Analysis of monoclonal Ab1 and Ab3 fine specificity

CaMBr1 is a tissue-specific and tumor-associated saccharidic epitope, defined by mAb MBr1 (Ab1), expressed on glycoconjugates of the human mammary carcinoma cell line MCF-7 and of normal and neoplastic mammary epithelial cells. An anti-anti-idiotypic monoclonal Ab3, 2G-3, identifying a human breast tumor associated antigen, was raised by using as immunogen a mouse anti-idiotypic monoclonal Ab2, A3B10, which behaves as the internal image of CaMBr1. mAb 2G-3, as well as MBr1, defines a saccharidic epitope on glycoconjugates extracted from MCF-7 cells and shows MBr1-like reactivity on normal and neoplastic-tissues. Experimental evidence, however, suggests that the fine immunoreactivity of the two antibodies is not identical, because MBr1 has a preferential reactivity with glycolipids and 2G-3 with glycoproteins. We suggest that a possible biologic explanation for our findings could reside in the nature of the immunogens used to raise the two mAb (glycolipid vs protein "internal image").     

Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling

We previously demonstrated that Fibroblast Growth Factor 10 (FGF10) and its receptor FGFR2b play a key role in controlling the very early stages of mammary gland development during embryogenesis [Mailleux, A.A., Spencer-Dene, B., Dillon, C., Ndiaye, D., Savona-Baron, C., Itoh, N., Kato, S., Dickson, C., Thiery, J.P., and Bellusci, S. (2002). Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development 129, 53-60. Veltmaat, J. M., Relaix, F., Le, L.T., Kratochwil, K., Sala, F.G., van Veelen, W., Rice, R., Spencer-Dene, B., Mailleux, A.A., Rice, D.P., Thiery, J.P., and Bellusci, S. (2006). Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. Development 133, 2325-35.]. However, the role of FGFR2b signaling in postnatal mammary gland development is still elusive. We show that FGF10 is expressed at high level throughout the adipose tissue in the mammary gland of young virgin female mice whereas its main receptor FGFR2 is found mostly in the epithelium. Using a rtTA transactivator/tetracycline promoter approach allowing inducible and reversible attenuation of the FGFR2b signaling throughout the adult mouse, we are now reporting that FGFR2b signaling is also critical during postnatal mammary gland development. Ubiquitous attenuation of FGFR2b signaling in the postnatal mouse for 6 weeks starting immediately after birth is not lethal and leads to minor defects in the animal. Upon dissection of the mammary glands, a 40% reduction in size compared to the WT control is observed. Further examination shows a rudimentary mammary epithelial tree with completely absent terminal end buds (TEBs), compared to a well-branched structure observed in wild type. Transplantation of mammary gland explants into cleared fat pad of wild type mouse recipients indicates that the observed abnormal branching results from defective FGFR2b signaling in the epithelium. We also demonstrate that this rudimentary tree reforms TEBs and resumes branching upon removal of doxycycline suggesting that the regenerative capacities of the mammary epithelial progenitor cells were still functional despite long-term inactivation of the FGFR2b pathway. At the cellular level, upon FGFR2b attenuation, we show an increase in apoptosis associated with a decrease in the proliferation of the mammary luminal epithelium. We conclude that during puberty, there is a differential requirement for FGFR2b signaling in ductal vs. TEBs epithelium. FGFR2b signaling is crucial for the survival and proliferation of the mammary luminal epithelial cells, but does not affect the regenerative potential of the mammary epithelial progenitor cells.     

In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.     

Mammary tumorigenesis in ApcMin/+ mice is enhanced by X irradiation with a characteristic age dependence

The Apc(Min/+) (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7-10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2-5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling.     

Molecular weight and subunit size of rabbit mammary-gland fatty acid synthetase.

1. The molecular weights of fatty acid synthetases isolated from lactating rabbit, rat, cow and goat mammary glands were estimated by sucrose gradient centrifugation and compared by chromatography on Sepharose 6B. 2. The values obtained for all four enzymes were in the same range (0.40 X 10(6)-0.55 X 10(6)) as that found for other mammalian and avian fatty acid synthetases. The molecular weight found for the rabbit mammary enzyme therefore differs from published values of approx. 0.9 X 10(6). 3. The molecular weights of the subunits of these four synthetases were 225000-242000. Again, the value for the rabbit mammary enzyme differs from published values.     

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.     

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K⁺ channel inhibitors Ba²⁺, quinine, and tetraethylammonium chloride, implicating K⁺ channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K⁺ channel, and also a dominant-negative NH₂-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K⁺ current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K⁺ secretion. potassium channel; volume regulation; mammary gland     

Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat

In this study, the Wistar-Kyoto (WKy) rat was genetically characterized for loci that modify susceptibility to mammary carcinogenesis. We used a genetic backcross between resistant WKy and susceptible Wistar-Furth (WF) rats as a panel for linkage mapping to genetically identify mammary carcinoma susceptibility (Mcs) loci underlying the resistance of the WKy rat. Rats were phenotyped for DMBA-induced mammary carcinomas and genotyped using microsatellite markers. To detect quantitative trait loci (QTL), we analyzed the genome scan data under both parametric and nonparametric distributional assumptions and used permutation tests to calculate significance thresholds. A generalized linear model analysis was also performed to test for interactions between significant QTL. This methodology was extended to identify interactions between the significant QTL and other genome locations. Chromosomes 5, 7, 10, and 14 were found to contain significant QTL, termed Mcs5, Mcs6, Mcs7, and Mcs8, respectively. The WKy alleles of Mcs5, -6, and -8 are associated with mammary carcinoma resistance; the WKy allele of Mcs7 is associated with an increased incidence of mammary cancer. In addition, we identified an interaction between Mcs8 and a region on chromosome 6 termed Mcsm1 (modifier of Mcs), which had no significant main effect on mammary cancer susceptibility in this genetic analysis.