Rat Mammary Epithelial Cells Research Articles

Synthesis of Gelatinases by Rat Mammary Epithelial and Myoepithelial Cell Lines

The synthesis of metalloproteinases capable of degrading the basement membrane is an important factor contributing to the involution of the mammary gland after the young are weaned. To investigate the cellular source of mammary gland metalloproteinases, we have studied the synthesis of gelatinases by rat mammary epithelial and myoepithelial cell lines immortalized with a temperature-sensitive T-antigen. At 39.5°C, the temperature at which the cells display increased differentiated characteristics, myoepithelial cells synthesize 40- to 160-fold greater amounts of three gelatinases ( M rs—72, 92, and 135 kDa) than epithelial cells. The gelatinases are preferentially secreted through the basolateral surface of myoepithelial cells. Growth of cells in the presence of a variety of growth factors and cytokines demonstrates the differential regulation of the synthesis of the 72- and 92-kDa gelatinases. These results suggest that the myoepithelial cell is a major source of mammary gland metalloproteinases.

Effects of epidermal growth factor on parathyroid hormone-related protein production by mammary epithelial cells

Parathyroid hormone-related protein (PTHrP) plays a prominent role in the pathogenesis of humoral hypercalcemia of malignancy. However, it is also expressed in various nonmalignant tissues, particularly during fetal organogenesis and tissue differentiation. Thus, PTHrP is synthesized in skin, placenta, and mammary gland during lactation. Little is known, however, about the regulation of PTHrP synthesis and release in nontumoral cells. We investigated the regulation of PTHrP production by epidermal growth factor (EGF), a factor of major importance in the development of lactating breast, in primary cultures of rat mammary epithelial cells. EGF stimulated the production of immunoreactive and bioactive PTHrP in a time- and concentration-dependent manner. A 12 h incubation with 10 ng/ml of EGF increased PTHrP production by 36.0 +/- 7.1% (n = 7 experiments, p < 0.01). This was accompanied by an increase in PTHrP mRNA steady-state levels. The production of PTHrP was stimulated by the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) by 82.9 +/- 9.7% (n = 4 experiments, p < 0.01). The effects of PMA and EGF were additive. The EGF-induced stimulation appeared to be independent of extracellular calcium concentration, prostaglandin, or cAMP synthesis, but may have involved tyrosine kinase-mediated mechanisms. These results indicate that EGF was capable of increasing the production of PTHrP by cultured mammary epithelial cells. They also suggest that factors activating the PKC pathway are involved in the upregulation of PTHrP expression in mammary epithelial cells.

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages

1. 1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([ 3H]-lysine, [ 14C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages. 2. 2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage. 3. 3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

Differentiational regulation and phosphorylation of the fatty acid-binding protein from rat mammary epithelial cells

From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 ± 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 ± 19 μg/ mg ( n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.

Phorbol 12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10(-6) M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10(-7) and 10(-6) M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10(-6) M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-alpha PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen.

Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar-like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:0-C14:0), using 14C-glucose, 14C-oleic acid or 14C-glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C-oleic acid labeling; 1.3 +/- 0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron-dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998 +/- 98% of control (P < 0.01) by the addition of phorbol 12-myristate 13-acetate (PMA) in combination with staurosporine, a protein kinase inhibitor. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13-fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5-fold increase). KT5720 (a cAMP-dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell-cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: 1) inhibition of a protein kinase and 2) a PKC-independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells.

Primary Culture of Flow Cytometry-Sorted Rat Mammary Epithelial Cell (RMEC) Subpopulations in a Reconstituted Basement Membrane, Matrigel

We studied the growth of rat mammary epithelial cell (RMEC) subpopulations cultured in Matrigel. Mammary glands were removed, minced, and enzymatically dispersed. Mammary organoids (ductal and endbud fragments) were collected, enzymatically monodispersed, filtered, and labeled with fluorescein isothiocyanate-peanut agglutinin (PNA) and phycoerythrin-anti-Thy-1.1 monoclonal antibody. By flow cytometry, we separated four RMEC subpopulations: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). The ultrastructural characteristics of sorted cells were examined by transmission electron microscopy. Thy-1.1+ cells appeared to be myoepithelial cells with actin filaments. PNA+, B-, and B+ cells were morphologically similar. Sorted RMEC subpopulations, unsorted mixed cells, and undispersed organoids were seeded in Matrigel and cultured in either a complete hormone medium (CHM) with 10% fetal bovine serum, progesterone, estradiol, hydrocortisone, insulin, and prolactin or a serum-free medium containing EGF, insulin, hydrocortisone, and human transferrin. Several types of colonies were observed: stellate, ductal, webbed, squamous, and lobuloductal colonies. At the immunocytochemical and electron micrograph levels, casein proteins were predominantly localized near the apical surfaces of the cells or in the lumina of ductal or lobuloductal colonies. Laminin and collagen IV were detected only in the basal lamina of ductal structures cultured in CHM. RMEC and subpopulations from immature virgin rats developed the capacity to synthesize and secrete lipid and casein when grown in Matrigel in serum medium or under defined serum-free conditions.

Estrous cycle modulation of O6-alkylguanine-DNA alkyltransferase expression in rat mammary epithelial cells.

N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor incidence and tumor number per rat, is directly correlated with an increase in the circulating level of estrogen(s) at the time of carcinogen administration and subsequent mammary epithelial O6-methylguanine content. We report that, expression of O6-alkyltransferase (AGT) is also regulated by reproductive hormones in a tissue specific manner. The level of mammary epithelial cell AGT activity on estrus (0.47 pmol/mg protein) and proestrus (0.32) was significantly higher than on metestrus (0.14) (P < 0.05, estrus vs. metestrus). However, no change was observed in liver AGT activity (0.52 pmol/mg protein). In contrast, the mean level of AGT protein was not significantly different between tumors from rats injected with MNU on different days of the estrous cycle. In conclusion, the different tumor biologies resulting from carcinogen injection on different days of the estrous cycle may be partially explained by variation in levels of DNA repair activity. However, the cells in the resulting tumors did not continue an obligatory differential expression of the AGT activity consistent with their stage of initiation.

Insulin like growth factor-I independence in rat mammary carcinoma cells: a dominant phenotype in somatic cell hybrid experiments

Normal rat mammary epithelial cells have an absolute requirement for insulin (IN) or insulin-like growth factor-I (IGF-I) for proliferation in serum-free medium. By contrast, serially transplantable rat mammary carcinoma (RMC) cells are IGF-I-independent for continuous growth in vitro. Previously, we demonstrated that IGF-I independence is not mediated by an autocrine loop. Therefore, experiments were carried out to determine if IGF-I independence behaves as a dominant or recessive phenotype in somatic cell hyhridization experiments. IGF-I-independent 1–9RMT cells were rendered hygromycin-resistant and IGF-I-dependent MCF-10A cells were rendered G418 resistant by infection with retroviral expression vectors. Cells of each line were co-incubated in 60 mm dishes and fused with polyethylene glycol. Hybrid cells were selected with media containing hygromycin and G418 in the presence or absence of IN. In three experiments, approximately the same number of colonies emerged in double selection media in the presence or absence of IN. Furthermore, colonies that emerged in IN-containing media, when subcultured, grew equally well in the presence or absence of IN. Thus, fusion of IGF-I-independent RMC cells with IGF-I-dependent human mammary epithelial cells yields hybrids that are IGF-I-independent for growth in serum-free medium.

Glycerophosphocholine and Phosphocholine Are the Major Choline Metabolites in Rat Milk

Choline is a constituent of cell membranes, surfactant and acetylcholine and is also a major source of methyl groups for the regeneration of methionine from homocysteine. Previous analyses of rat, human and bovine milk measured only choline, phosphatidylcholine and sphingomyelin. Choline-containing compounds in milk from rats lactating for 15 d were measured by HPLC and gas chromatograph-mass spectrometry. In addition to the previously reported choline metabolites, substantial concentrations of glycerophosphocholine (3.7 mmol/L) and phosphocholine (653 mumol/L) were also detected. At 1 h after oral administration of [methyl-14C]choline to lactating rats, the major labeled metabolites were phosphocholine (91% of label in milk) and betaine (9%). Twenty-four hours after the dose, glycerophosphocholine was the major labeled metabolite (69% of label in milk). Rat mammary epithelial cells, in primary culture, synthesized and secreted phosphatidylcholine, phosphocholine, glycerophosphocholine and betaine. Thus, the mammary gland was able to synthesize the choline metabolites found in milk, but these metabolites may not be derived exclusively from uptake from maternal blood. We have established that the total choline concentration in rat milk is sevenfold higher than previously reported, with > 80% present as glycerophosphocholine and phosphocholine.

Casein Accumulation by Rat Mammary Epithelial Cells Grown within a Reconstituted Basement Membrane Is Modulated by Fatty Acids in a Hormone- and Time-Dependent Manner

The mammary gland is under complex regulation involving the participation of hormones, growth factors, and stromal components, including lipids. Our laboratory has developed a unique primary culture system that allows undifferentiated mammary epithelial cells from immature virgin rats to proliferate and differentiate to an extent equivalent to the lactating mammary gland. Using this model system we have examined the effects of the unsaturated fatty acids oleate and linoleate on mammary epithelial cell proliferation as well as both morphological and functional differentiation. Neither fatty acid showed any effect on cell proliferation whether added to cells in the presence of optimal serum-free medium or under suboptimal conditions of epidermal growth factor (EGF) and prolactin. Morphological differentiation also was not affected by fatty acid addition under either optimal or suboptimal conditions, although a decrease was observed when medium depleted in EGF and prolactin was compared to optimal medium. The notable finding in this study was that both oleate and linoleate modulated functional differentiation, as assessed by casein accumulation, in a time- and hormone-dependent manner. At early times in culture, casein levels were stimulated by both oleate and linoleate; this effect was most dramatic under suboptimal conditions of prolactin and EGF. In marked contrast, however, linoleate decreased casein levels by approximately 50% in optimal medium, at all concentrations tested, after at least 7 days in culture. This decrease was also observed in suboptimal medium, although the concentration of EGF and prolactin influenced the extent of the reduction. Although the mechanism is currently unknown, it is tempting to speculate that the cellular and biochemical events that result in linoleate-induced inhibition of functional differentiation may also be involved in the tumor-enhancing properties of this fatty acid.

Production of the metastatic phenotype by DNA transfection in a rat mammary model.

A syngeneic, immunocompetent rat mammary model has been employed to investigate the molecular basis of the metastatic phenotype. Transfection of the benign rat mammary epithelial cell line Rama 37 with the gene for p9Ka; a small, rat, calcium-binding protein or DNA from metastatic human cell lines derived from a primary breast carcinoma or a pleural effusion yields transfectants with metastatic capabilities. Transfection of DNA from a benign human mammary cell line, a plasmid containing a cDNA for the human basic fibroblast growth factor (bFGF) gene, or the oncogene EJ-ras-1, fails to yield any transfectants expressing the metastatic phenotype.

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Characterization of Rat Mammary Epithelial Cell Subpopulations by Peanut Lectin and Anti-THY-1.1 Antibody and Study of Flow-Sorted Cells in Vivo

A cell separation method was developed for studies of the growth kinetics of rat mammary epithelial cell (RMEC) subpopulations in grafts and in culture in vitro . By flow cytometry of RMEC stained with fluorescein isothiocyanate-peanut agglutinin and phycoerythrin-anti-Thy-1.1 monoclonal antibody, we could distinguish four cell subpopulations from primary cultures of 7- to 8-week-old F344 female rat mammary glands: both negative (B-), PNA+, Thy-1.1+, and both positive (B+). We studied the growth patterns of these subpopulations in vitro for 1 to 14 days in complete hormone medium (CHM) containing 10% fetal bovine serum and prolactin, 17β-estradiol, cortisol, progesterone, and insulin. The fractions of PNA+ and B- cells steadily decreased with time in culture. The fraction of Thy-1.1+ cells steadily increased with time in culture. There were small numbers of B+ cells. We grafted RMEC in hyperprolactinemic recipient rats. The mean numbers of transplanted cells required to produce at least one alveolar unit in 50% of the graft sites (AD50 values) are inversely related to the clonegenie fractions. AD50s of RMEC are as follows: unsorted mammary cells cultured in CHM for 1 to 4 days, ∼220; unsorted cells cultured in CHM for 7 days, ∼550; sorted PNA+ RMEC from outgrowths of 3-day cultures, ∼82; B+, ∼350; B-, ∼545; Thy-1.1+, ∼9372. We conclude that the PNA+ cell subpopulation includes most of the clonegenic cells. Thy-1.1+ cells appear to he terminally differentiated; they are likely to be myoepithelial cells.

Low molecular mass GTP-binding proteins are secreted from mammary epithelial cells in association with lipid globules

Secretion of milk lipid globules is achieved through encapsulation of triacylglycerol-rich lipid droplets in a specialized region of apical plasma membrane of mammary epithelial cells. A class of low molecular mass GTP-binding proteins were associated tightly with the lipid globule membrane, and these proteins appeared to change from peripheral to integral membrane proteins during intracellular growth and transit of lipid globule precursors. Inclusion of GTP or GTPγS in incubation medium stimulated secretion of lipids from primary cultures of permeabilized rat mammary epithelial cells. Six polypeptides with molecular masses between 28 and 21 kDa were detected by ability to bind GTPγS following separation of lipid-globule-associated proteins by SDS-PAGE and transblotting onto nitrocellulose. That all of these polypeptides were distinct immunologically from the archetype ras was evident from lack of immunoreactivity with p 21ras G-protein monoclonal antibody in Western blots. This monoclonal antibody bound to a 23 kDa polypeptide of lipid droplets that was not detected with the GTPγS binding assay. A 25 kDa component of milk lipid globules was a potent substrate for ADP-ribosylation by botulinum toxin C3, but cholera toxin was much less effective, suggesting that this component may belong to the rac class of G-proteins. The 21 kDa component was related immunologically to ADP ribosylation factor.

Ectopic production of heparin-binding growth factors and receptors for basic fibroblast growth factor by rat mammary epithelial cell lines derived from malignant metastatic tumours.

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10(6) cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (KD 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (KD 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (KD 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.

Hydrocortisone induces the synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells.

The effects of lactogenic hormones on protein secretion by 25.5-G4.2.3 cells, a rat mammary myoepithelial cell line immortalised with a temperature-sensitive T-antigen, were investigated. Insulin, prolactin, estradiol and progesterone had no effect but hydrocortisone induced the secretion of two proteins with molecular masses of 175 kDa (p175) and 146 kDa (p146), 10-30-fold and 5-fold respectively. The induction of p175 and p146 synthesis by hydrocortisone was greater at 39.5 degrees C than at 33 degrees C reflecting the increased differentiation of 25.5-G4.2.3 cells at the higher temperature. Rat mammary epithelial cells did not synthesise p175. After addition of hydrocortisone to 25.5-G4.2.3 cells, there was a lag phase of 10 h before the synthesis of p175 was induced. Half-maximal induction of p175 synthesis required a hydrocortisone concentration of 0.5 microM. p175 was identified as alpha 2-macroglobulin by N-terminal amino-acid sequence determination and immunoprecipitation with a specific antibody. Hydrocortisone induced a 5-kb alpha 2-macroglobulin-specific mRNA transcript in 25.5-G4.2.3 cells. Myoepithelial cells are responsible for synthesising the basement membrane around the rapidly expanding mammary alveoli during pregnancy. Myoepithelial cells also secrete metalloproteinases which are probably involved in turnover of the basement membrane. We suggest that increased levels of hydrocortisone during pregnancy induce the synthesis of alpha 2-macroglobulin, which is believed to be a potent inhibitor of metalloproteinases, by rat mammary myoepithelial cells to reduce proteolytic degradation of the basement membrane.

Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer

The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV approximately CMV approximately SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.

Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells

Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells

Mitogenic effect of a factor from rat somatomammotrophs on mammary epithelial cells.

We have previously established a somatomammotroph cell line (rPC0) derived from normal rat pituitary that secretes GH and PRL. In this study we report that conditioned medium from rPC0 cells (CM-rPC0) exhibited a stimulatory effect on the growth of rat mammary epithelial cells in culture. Fractionation of CM-rPC0 revealed that the growth-promoting activity of CM-rPC0 was associated with a fraction eluting from a diethylaminoethyl-Sephacel column at 0.5 M NaCl. The growth-promoting activity of this fraction was abolished by trypsin and was resistant to dithiothreitol treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the predominant components of the 0.5-M NaCl fraction were proteins migrating at the 14-18K region of the gel. The 0.5-M NaCl fraction did not contain immunodetectable GH or PRL. Further fractionation of CM-rPC0 on a heparin-agarose column showed that the growth-promoting activity eluted at 0.9 M NaCl. The two predominant proteins in this fraction had apparent mol wt of 14.5K and 18.2K. Based on the structural and biological properties, several known hormones and growth factors were excluded from consideration as potential candidates responsible for the growth-promoting effect of the 14-18K proteins. It is postulated that this group of 14-18K proteins contains a new factor(s) that affects the growth of mammary epithelial cells.