PMC42, a novel model for the differentiated human breast

Cultured human breast carcinoma cell lines are important models for investigating the pathogenesis of breast cancer. Their use, however, is limited because of loss of expression of breast-specific markers and the development of a dedifferentiated phenotype after continuous culture. PMC42 is a unique human breast carcinoma line, previously shown to express secretory and myoepithelial markers. We have induced PMC42 cells to form hollow organoids in culture, similar to in vivo breast structures, using a combination of hormones including estrogen, progesterone, dexamethasone, insulin, and prolactin in combination with a permeable extracellular matrix. The organoids comprised polarized cells located around a central lumen. Expression of β-casein was demonstrated in cells within organoids using reverse transcriptase-polymerase chain reaction, Western blot analysis, and confocal immunofluorescence. In this in vitro system, milk-specific gene expression was induced through hormone and matrix interactions which may be similar to those operating in vivo. PMC42 is a novel model for investigations into the molecular mechanisms of carcinogenesis and differentiation in the human breast.

Copper is taken up efficiently from albumin and α2-macroglobulin by cultured human cells by more than one mechanism

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human  albumin and α2-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3–6 µM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from   α2-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65–80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100–500 µM) inhibited copper uptake from albumin by 20–30% in both cell types and that from {alpha}2-macroglobulin by 0–30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms.α2-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background
Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.


Methods
We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.


Results
We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.


Conclusion
We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Extracellular matrix induces formation of organoids and changes in cell surface morphology in cultured human breast carcinoma cells PMC42-LA

In the lactating breast, the development of secretory alveoli consisting of differentiated cells arranged around a central lumen is dependent on signals from the extracellular environment of the cells. There are few cell lines that model this process. We previously showed that the human breast carcinoma line PMC42-LA can be induced to form organoids, reminiscent of secretory alveoli found in the lactating human breast. In this report, we used high-resolution scanning electron microscopy to show that the formation of organoids is accompanied by development of cell surface microvilli. Extracellular matrix-induced formation of microvilli occurred on the internal and external surfaces of cells in the organoids and not on surfaces in contact with the extracellular matrix. Organoid formation of PMC42-LA cells induced a rearrangement of the extracellular matrix, seen in the form of radiating fibers from the organoids. In summary, there is an interaction between PMC42-LA cells and the underlying extracellular matrix, which leads to the formation of polarized cells with well-developed microvilli. This is accompanied by organization of the extracellular matrix. PMC42-LA is a relevant model of the human breast for investigations into cell-cell and cell-matrix interactions.

Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

Introduction Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). Results Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. Conclusions This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Defining the E-Cadherin repressor interactome in epithelial-mesenchymal transition : the PMC42 model as a case study

Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells

Zinc is an essential trace element required by all living organisms. An adequate supply of zinc is particularly important in the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. The mechanisms by which zinc becomes a constituent of breast milk have not been elucidated. The function of the zinc transporter ZnT4 in the transport of zinc into milk during lactation was previously demonstrated by studies of a mouse mutant, the ‘lethal milk’ mouse, where a mutation in the ZnT4 gene decreased the transport of zinc into milk. In the present study, we have investigated the expression of the human orthologue of ZnT4 (hZnT4) in the human breast. We detected hZnT4 mRNA expression in the tissue from the resting and lactating human breast, using reverse-transcriptase PCR. Western-blot analysis using antibodies to peptide sequences of hZnT4 detected a major band of the predicted size of 47 kDa and a minor band of 77 kDa, in extracts from the resting and lactating breast tissues. There was no difference in the hZnT4 expression levels between lactating and resting breasts. The hZnT4 protein was present in the luminal cells of the ducts and alveoli where it had a granular distribution. A cultured human breast epithelial cell line PMC42 was used to investigate the subcellular distribution of hZnT4 and this showed a granular label throughout the cytoplasm, consistent with a vesicular localization. The presence of zinc-containing intracellular vesicles was demonstrated by using the zinc-specific fluorphore Zinquin (ethyl-[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate). Double labelling indicated that there was no obvious overlap between Zinquin and the hZnT4 protein, suggesting that hZnT4 was not directly involved in the transport of zinc into vesicles. We detected expression of two other members of the hZnT family, hZnT1 and hZnT3, in human breast epithelial cells. We conclude that hZnT4 is constitutively expressed in the human breast and may be one of the several members of the ZnT family involved in the transport of zinc into milk.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

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 (⁶⁴Cu) 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.