Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation.

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic "cobblestone" morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in beta-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) alpha s- and beta-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells

Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

The Membrane-Associated Form of αs1-Casein Interacts with Cholesterol-Rich Detergent-Resistant Microdomains

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α_s1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α_s1-casein in rat mammary epithelial cells. Using metabolic labelling we show that α_s1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α_s1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α_s1-casein. These experiments reveal that the insolubility of α_s1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α_s1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.     

In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).     

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins.

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.     

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.     

Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis

Summary Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 ± 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulinlike growth factor-I, and somatotropin + insulinlike growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G₀G₁ phase was increased by: (a) insulinlike growth factor-I in explant cells; (b) somatotropin, insulinlike growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulinlike growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G₀G₁ or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more β-casein than primary cells but secretion trends for β-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.     

Pten Regulates Development and Lactation in the Mammary Glands of Dairy Cows

Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of β-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for β-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPARγ, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of β-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased β-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.     

Studies on human alpha(s)- and kappa-casein fractions and human caseinoglycomacropeptide

1. Fractions have been obtained from human whole casein closely resembling the α_s- and κ-fractions of cow casein. 2. The α_s-fraction (human α_s-casein) is calcium-sensitive, heterogeneous in zone analysis and inert towards rennin. 3. The κ-fraction (human κ-casein) is calcium-insensitive, heterogeneous in zone analysis, and forms a soluble glycopeptide when acted upon by rennin. 4. Human κ-casein stabilizes human α_s-casein in the presence of Ca²⁺ ions. 5. The glycopeptides released by rennin from human casein and from cow casein have been compared. There are important differences in both the peptide and non-peptide structures of the two compounds. 6. In both human and bovine glycopeptides some of the carbohydrate residues are joined to the peptide by O-glycosidic links with threonine, and possibly with serine.     

RECOMBINANT HUMAN INTERLEUKIN-8, BUT NOT HUMAN INTERLEUKIN-1β, INDUCES BOVINE NEUTROPHIL MIGRATION IN AN IN VITRO CO-CULTURE SYSTEM

A co-culture system was established by culturing a bovine mammary epithelial cell line (MAC-T) and a bovine aortic endothelial cell line on calf tail collagen pre-coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin-1β (rHIL-1β) and interleukin-8 (rHIL-8) on bovine neutrophil migration was investigated using this system. rHIL-8 stimulated bovine neutrophil migration in a dose-dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100 ng/ml of rHIL-8 was used. On the other hand, rHIL-1β at concentrations up to 100 ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre-incubation with 5 ng/ml of rHIL-1β in the co-culture system for 4 or 24 h failed to have any effect. These results suggest that IL-8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.     

Overexpression of ovine insulin-like growth factor-I stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells.

To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-I under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I peptides.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.     

The separation of major components of the casein of bovine milk by electrophoresis in a density gradient

1. Zonal electrophoresis in a column stabilized by a density gradient has been applied to the small-scale fractionation of the proteins of the casein complex of cow's milk. 2. The α_s- and β-fractions from the milk of individual Ayrshire cows have been shown to behave as single homogeneous proteins on electrophoresis at two pH values in starch gels. 3. The α_s-fraction has been found to be indistinguishable from α_s-casein prepared by Ca²⁺ fractionation of the same milk samples. 4. On the evidence of their electrophoretic behaviour in starch gels and their elementary analyses, α₁- and α_s-casein are concluded to be substantially the same protein.     

A Role for Interleukin-1 Alpha in the 1,25 Dihydroxyvitamin D3 Response in Mammary Epithelial Cells

Breast cancer is the most common non-cutaneous malignancy in American women, and better preventative strategies are needed. Epidemiological and laboratory studies point to vitamin D₃ as a promising chemopreventative agent for breast cancer. Vitamin D₃ metabolites induce anti-proliferative effects in breast cancer cells in vitro and in vivo, but few studies have investigated their effects in normal mammary epithelial cells. We hypothesized that 1,25(OH)₂D₃, the metabolically active form of vitamin D₃, is growth suppressive in normal mouse mammary epithelial cells. In addition, we have previously established a role for the cytokine interleukin-1 alpha (IL1α) in the anti-proliferative effects of 1,25(OH)₂D₃ in normal prostate cells, and so we hypothesized that IL1α is involved in the 1,25(OH)₂D₃ response in mammary cells. Evaluation of cell viability, clonogenicity, senescence, and induction of cell cycle regulators p21 and p27 supported an anti-proliferative role for 1,25(OH)₂D₃ in mammary epithelial cells. Furthermore, 1,25(OH)₂D₃ increased the intracellular expression of IL1α, which was necessary for the anti-proliferative effects of 1,25(OH)₂D₃ in mammary cells. Together, these findings support the chemopreventative potential of vitamin D₃ in the mammary gland and present a role for IL1α in regulation of mammary cell proliferation by 1,25(OH)₂D₃.     

Possible involvement of prolactin in the synthesis of lactoferrin in bovine mammary epithelial cells

Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.