Rat Mammary Epithelial Cells Research Articles

Effect of trypsin in the presence or absence of Ca2+ on tight junction in an epithelial cell line

Recent studies indicate that adhesion between cells is mediated by at least two different surface sites, one Ca2+-dependent and one Ca2+-independent. The first is highly sensitive to trypsin in the absence of Ca2+ but resistant in the presence of Ca2+, whereas inactivation of the second requires higher trypsin concentrations and Ca2+ has no effect on proteolysis.Tight junctions (TJ) in vertebrate epithelia provide a transepithelial permeability barrier, help to maintain cell polarity, and also are strongly adhesive. Trypsin and other proteases are often used in cell dissociation protocols but are known in several epithelia to induce TJ proliferation. Our previous study of LA7, a rat mammary epithelial cell line, showed as demonstrated by others that withdrawal of Ca2+ leads to disruption of TJ. The effect of trypsin on LA7 cells in the absence or presence of Ca2+ was studied to determine whether TJ are related to the Ca2+-dependent mechanism of cell adhesion in epithelia.

Induction of differentiation in a rat mammary epithelial stem cell line by dimethyl sulphoxide and mammotrophic hormones.

Rama 25 is a clonal epithelial cell line derived from a dimethylbenzanthracene-induced rat mammary adenocarcinoma. In the presence of the mammotrophic hormones, insulin, hydrocortisone, estrogen and prolactin, Rama 25 produces small amounts of casein and forms domes at a low rate. The rates of both these processes can be greatly increased by the addition of dimethyl sulphoxide or hexamethylenebisacetamide which are also known to induce the differentiation of Friend erythroleukemia cells. Other compounds which stimulate the differentiation of Rama 25 cells include linoleic acid and 6-thioguanine. The intracellular pathways triggering changes in the two markers of differentiation are partially separable using different combinations of hormones, prolactin and hydrocortisone being the most important for the production of casein and the formation of domes respectively. The kinetics of differentiation, as judged by the appearance of these two markers, are characterised by two phases, a fixed period of 8 h (lag phase), the length of which is independent of the dimethyl sulphoxide concentration and a second phase where their rates are dependent on the concentration of dimethyl sulphoxide. Rama 25 cells do not become committed to differentiate during this lag phase but increasing numbers of cells do so after this period. We suggest that the differentiation processes occur in two stages. The first stage, involving the inducer, commits Rama 25 cells to a new differentiated state. The second stage, involving the hormones, modulates the expression of different markers of this state. Both casein production and dome formation can be blocked by inhibitors of DNA synthesis and show reciprocal changes with the rates of cellular DNA synthesis. Thus, in its hormonal and DNA synthetic requirements for differentiation, Rama 25 cells appear to resemble some of the mammary epithelial cells of mature virgin rats.

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.

In vitro metabolism of 7,12-dimethylbenz[a]anthracene in mammary epithelial cells from rats with different susceptibilities to carcinogenesis.

These in vitro metabolism studies of 7,12-dimethylbenz[a]anthracene (DMBA) by rat mammary epithelial cells were initiated to determine whether differences in the incidence of DMBA-induced mammary carcinomas observed between young virgin, old virgin and parous Sprague-Dawley rats can be explained through variations in their ability to metabolize DMBA. The results show that: (a) qualitatively, the metabolic profiles produced by epithelial cells from the 3 groups of rats were similar; (b) the level of metabolism with cells from young virgin and parous rats was similar but was 1.5-2.0 times higher than that obtained with cells from old virgin rats; c) the major ethyl acetate-soluble metabolite detected by h.p.l.c. from cells of old virgin and parous rats was the trans-8,9-dihydrodiol (50-65% of total metabolites), whereas the majority of the radioactivity following incubation with cells from young virgin rats co-eluted with very polar metabolites (65% of total metabolites); and (d) the amount of phenolic metabolites produced by young virgin rat cells was higher than in old virgin or parous rat cells. The data suggest that the susceptibility of young virgin rat mammary cells to DMBA may be due in part to the conversion of a greater percentage of DMBA to phenolic compounds and to water- and ethyl acetate-soluble polar metabolites, and event known to influence the susceptibility of cultured cells to the cytotoxic effects of DMBA.

The kinetics of in situ repair in rat mammary cells.

The kinetics of in situ repair (ISR) was investigated in normal rat mammary epithelial cells exposed in vivo to 8 Gy 137 Cs gamma rays. ISR as measured by transplantation assay was completed by 4 hours after exposure. Although ISR in the three normal epithelial cell types thus far tested differs from repair of potentially lethal damage in tumour cells and cultured fibroblasts in that it affects the shoulder and not the slope of the survival curve, the current study shows that the time course of ISR resembles that for repair of potentially lethal damage in both tumours and fibroblasts.

Isolation and characterization of a growth factor (embryonin) from bovine fetuin which resembles alpha 2-macroglobulin.

A serum-free hormone-supplemented medium which was previously formulated for the growth of mouse embryonal carcinoma (EC) cells and rat mammary epithelial (REM) cells required the presence of crude bovine fetuin as a medium supplement for maintaining cell growth. This requirement could not be replaced by purified fetuin preparations. The major growth-promoting activity (embryonin) in the crude fetuin preparation has been purified to near homogeneity by carboxymethylcellulose chromatography and high performance gel filtration chromatography. Purified embryonin, 2 to 4 micrograms/ml, is able to stimulate the growth of mouse EC cells in a serum-free hormone-supplemented medium to a level that is achieved with 0.5 to 1 mg/ml of the crude fetuin preparation. The biological activity resides in a high molecular weight glycoprotein (Mr = 270,000). Three polypeptide chains are observed following reduction, a major polypeptide (Mr = 185,000) and two minor chains (Mr = 116,000 to 120,000 and 68,000). Embryonin differs from pure fetuin in molecular weight, isoelectric point, amino acid composition, and immunological reactivity. However, embryonin is similar to human alpha 2-macroglobulin (alpha 2M) in these physicochemical and immunological properties. Resemblance to alpha 2M is also indicated by the observation that alpha 2M used over the same protein concentration range as embryonin produces a comparable stimulation of EC cell growth in the absence of serum. In addition, embryonin and alpha 2M produce a 2- to 10-fold differential stimulation of collagen production in cultures of normal rat kidney and RME cells, a response which may be linked to the growth-promoting activity of these two proteins.

Control of protein synthesis in cuboidal rat mammary epithelial cells in culture. Changes in gene expression accompany the formation of elongated cells.

The cuboidal rat mammary tumour cell line, Rama 25, spontaneously converts to elongated cells during culture. The polypeptides synthesised by the two morphological forms are compared by two-dimensional polyacrylamide gel electrophoresis. In addition to major changes in the intermediate filament proteins, there are two abundant acidic polypeptides of molecular weights 9000 and 15500, which are synthesised by the elongated cells but which are not detectable in the cuboidal cells. When mRNA preparations isolated from the cuboidal and elongated cells are translated in the reticulocyte lysate, the patterns of radioactive products resemble the patterns of polypeptides synthesised in the intact cells. Translation products corresponding to the 9000-Mr and 15500-Mr polypeptides are synthesised when mRNA from elongated cells is translated, but are not synthesised when mRNA from cuboidal cells is translated. Evidence is presented that the appearance of these two proteins represents a change in gene expression between the two cell types and that control is exerted prior to mRNA translation.

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Differential staining techniques based on the incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA permit the unequivocal identification of metaphrase cells which have replicated once, twice and three or more times. This technique is used to analyze the cell replication kinetics of mammary epithelial cells from young virgin rats in culture. Comparison is made between these in vitro observations and expected results derived from in vivo determination of labelling index, growth fraction, cell cycle length and distribution for the different structures and cell types of the rat mammary gland. The correlation coefficient for the comparison between in vivo and in vitro results is 0.84 suggesting that the heterogeneity of cell population and replication kinetics of the rat mammary gland is preserved when cells are placed in culture. Furthermore, the BrdU incorporation system for the analysis of cell replication kinetics is accurate and acceptable for the study of rat mammary epithelial cell kinetics.

Localization of thioesterase II, the chain-length regulatory enzyme of milk fatty acid synthesis, in rat mammary gland epithelial cells.

Two approaches were used to establish the intercellular distribution of fatty acid synthetase and thioesterase II in the lactating rat mammary gland. Thioesterase II is the chain-length regulatory enzyme in the biosynthesis of the medium-chain fatty acids characteristic of milk fat. Using immunohistochemical techniques, immunoreactive fatty acid synthetase was found in both mammary adipocytes and epithelial cells; in contrast, immunoreactive epithelial cells were isolated from lactating rat mammary glands after digestion with collagenase and thermolysin, and their lipogenic activity was studied using isotopically labelled acetate. Consistent with the immunohistochemical data, adipocytes synthesized exclusively long-chain fatty acids whereas epithelial cells synthesized predominantly chain fatty acids. The results indicate that the capacity for synthesis of medium-chain fatty acids is a unique property of the epithelial cell component of the mammary gland.

Isolation and characterization of a serially cultivated, neoplastic, epithelial cell line from the N-nitrosomethylurea induced rat mammary adenocarcinoma.

A new in vitro model for human breast cancer is described. Derived from an N-nitrosomethylurea (NMU) induced rat mammary adenocarcinoma, this serially cultivated cell line has been demonstrated, by a variety of criteria, to be an authentic neoplastic, rat mammary epithelial cell line. The criteria used include morphological and growth characteristics; the presence of specific cell surface antigens; steroid hormone receptors; hormone responsiveness; casein production; karyotype and isoenzyme profile analysis; anchorage independent growth and oncogenicity. Inasmuch as the NMU cell line possesses high concentrations of glucocorticoid and androgen receptors, it may provide a useful model for study of the action of these hormones in human breast cancer. In addition, the NMU line may serve as a valuable in vitro model in which to assess the effects of a variety of endogenous and exogenous agents known to influence mammary tumor growth in vivo, including drugs, nutrients, and growth factors.

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.

Reconstitution of branching tubules from two cloned mammary cell types in culture

Rat mammary epithelial tumor cells from the line Rama 25 can grow into three-dimensional, multicellular structures when cultured on floating collagen gels. These structures include branching tubules reminiscent of ducts in glands, and the production of the tubules is studied here as a model of glandular morphogenesis. The cell line contains cells of two types which can be cloned and grown separately. Tubules are formed by neither cell type alone, but by combinations. The behavior of the two cell types suggests a mechanism for the growth of glands.

Formation and removal of 7,12-dimethylbenz[a]anthracene--nucleic acid adducts in rat mammary epithelial cells with different susceptibility to carcinogenesis.

Formation and removal of 7,12-dimethylbenz[a]anthracene--nucleic acid adducts in rat mammary epithelial cells with different susceptibility to carcinogenesis.

Casein kinase in cytosol of rat and mouse mammary epithelial cells isolated from histone kinase by MgCl2 treatment and influence of pregnancy and lactation on the enzyme activity.

Rat mammary glands contain cyclic AMP-independent casein kinase and cyclic AMP-dependent histone kinase. The former was easily isolated from cyclic AMP-dependent histone kinase by MgCl2 treatment. Mammary casein kinase was not activated by cyclic nucleotides, and Mg++ and ATP were required for activation. The specific activity of casein kinase in cytosol of rat mammary epithelial cells increased 2 to 3-fold during pregnancy and lactation. Cytosol of mouse mammary epithelial cells also contained cyclic AMP-independent casein kinase, and the activity of this enzyme was about three times that of the Golgi fraction.

Differential response to growth factor by rat mammary epithelium plated on different collagen substrata in serum-free medium.

Primary cultures of rat mammary epithelial cells proliferate and synthesize basement membrane collagen (type IV collagen) in a serum-free medium supplemented with epidermal growth factor (EGF), hydrocortisone or dexamethasone, insulin, transferrin, and Pedersen fetuin. The growth response of the cells to EGF and glucocorticoids but not to insulin or transferrin varies depending on the substratum on which the cells are plated. Cell growth is 4 times more sensitive to omission of EGF or glucocorticoid on type I collagen or plastic substratum than on type IV collagen substratum. The mechanism by which these two growth factors differentially affect cell growth appears to be linked to an increase in type IV collagen synthesis and a stabilization of secreted type IV collagen in the extracellular matrix. Glucocorticoids suppress the elaboration of type IV collagenolytic activity by the cells whereas EGF stimulates amino acid incorporation into type IV collagen. The results suggest that EGF and glucocorticoids affect mammary epithelial cell growth by facilitating the accumulation of the appropriate cell substratum.

Aryl Hydrocarbon Hydroxylase and Epoxide Hydratase Activities: Age Effects in Mammary Epithelial Cells of Sprague-Dawley Rats<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

By use of techniques developed for rat mammary epithelial cell isolation, studies were conducted to characterize aryl hydrocarbon hydroxylase (AHH) and epoxide hydratase (EH) activities and to determine whether changes in the enzyme activities might contribute to the age-dependent changes in polycyclic aromatic hydrocarbon (PAH)-induced tumorigenesis in the rat mammary gland. Constitutive AHH and EH activities in rat mammary epithelial cell homogenates were substantially lower than those measured in liver (9,500×g) supernatant. The administration of 3-methylcholanthrene (MCA) or 7,12-dimethylbenz[a]anthracene increased mammary epithelial AHH activity 93- and 44-fold, respectively, whereas hepatic AHH levels rose 17- and 6-fold. Neither inducer had any effect on constitutive EH activity in either tissue. The addition (0.1 mM) of 7,8-benzoflavone (7,8-BF), SKF 525-A, or metyrapone to homogenates of mammary epithelial cells from MCA-treated rats inhibited AHH activity 93, 48, and 22%, respectively. In contrast, 7,8-BF addition decreased rat liver AHH activity 83%, whereas SKF 525-A and metyrapone had little effect. EH activities in rat liver and mammary epithelial cells were reduced with addition of 1,1,1-trichloro-2-propene oxide and cyclohexene oxide; however, trans-stilbene oxide had no effect in either tissue preparation. Additional studies determined the age-dependent changes in rat mammary epithelial AHH and EH activities. Constitutive AHH activity remained unchanged in rats between 40 and 100 days of age. In contrast, mammary epithelial EH activity was highest in 40-day-old Sprague-Dawley rats, declined in rats between 40 and 60 days of age, and remained constant in rats between 60 and 100 days of age. The level of MCA-induced AHH activity in rat mammary epithelial cell homogenates was age-dependent. MCA administration induced constitutive AHH activity 85- and 74-fold in mammary epithelial cells isolated from 50- and 60-day-old Sprague-Dawley females. The magnitude of mammary epithelial AHH induction was significantly lower in both younger and older animals. The results demonstrated PAH-metabolizing enzymes in rat mammary epithelial cells and suggested that the age-dependent incidence of mammary tumorigenesis may be attributed in part to the age-dependent inducibility of AHH activity.

Role of microtubules in milk secretion ? Action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells

Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.

Binding of Peanut Lectin to Breast Epithelium, Human Carcinomas, and a Cultured Rat Mammary Stem Cell: Use of the Lectin as a Marker of Mammary Differentiation

We investigated the binding of fluorescence-labeled peanut agglutinin (PNA) to breast epithelium. Specific binding of PNA to the mammary glands of female Sprague-Dawley rats increased as the gland matured. Sexually immature rats showed relatively little fluorescence, but this increased in mature and pregnant animals. A maximum was reached in lactating rats in which significant labeling of material within the lumen was observed. PNA was bound exclusively to the epithelial and not the myoepithelial or mesenchymal cells. In tissue culture, a rat mammary epithelial stem cell line, which can be stimulated to differentiate to alveolus-like secretory or myoepithelial cells, showed evidence of PNA binding only on the secretory cells and not on unstimulated or myoepithelial cells. Fibroblast cultures also failed to show significant binding of PNA. Receptor sites on the secretory cells were masked mainly by sialic acid. Human breast sections, like those of the rat, showed fluorescent labeling at the apical region of the epithelial cells; this labeling increased if the tissue had prior treatment with neuraminidase. Breast carcinomas that were morphologically differentiated showed more labeling with PNA than did undifferentiated tumors, which often had weak or sometimes negative labeling. When significant fluorescence was observed, it was localized mainly in the cytoplasm. By contrast, labeling was restricted to the cell periphery in differentiated carcinomas. The use of PNA as a marker for breast epithelial cell differentiation is therefore proposed.

Characteristics of aryl hydrocarbon hydroxylase activity in rat mammary epithlial cells grown in primary culture

Rat mammary epithelial cells grown in primary culture contain the microsomal enzyme, aryl hydrocarbon (benzo[ a]pyrene) hydroxylase (AHH), which catalyses the oxidative conversion of polycyclic aromatic hydrocarbons (PAH) to more polar derivatives. Constitutive AHH activity, measured with an established fluorometric method, was 46 pmol/mg protein/h in homogenates of rat mammary epithelial cells after 5 days in culture. The addition of dimethylbenz[ a]anthracene (DMBA), benz[ a]anthracene (BA), or 3-methylcholanthrene (3-MC) to the cell culture medium increased AHH activity 5.3-, 4.7- and 2.4-fold, respectively. Kinetic studies revealed that maximal hydroxylase induction occurred 16 h after 1 μM DMBA was added to the culture medium. The decay of the DMBA-induced hydroxylase was biphasic: one component had a t 1 2 of 15–30 min and another a t 1 2 of 4 h. Norepinephrine, 17β-estradiol and 5,6-benzoflavone also increased AHH activity in mammary epithelial cells in vitro, however, sodium phenobarbital had no effect. Fetal bovine serum (FBS), previously shown to be a potent in vitro inducer of AHH activity, had no effect on either constitutive or DMBA-induced mammary epithelial hydroxylase activities following treatment with 1% activated charcoal. Metyrapone and 7,8-benzoflavone, inhibitors of microsomal mixed function oxidase activity, reduced both constitutive and DMBA-induced AHH activities when added to homogenates of untreated and DMBA-treated mammary epithelial cells. The addition of 7,8-benzoflavone reduced both constitutive and DMBA-induced hydroxylase activities by approx. 80%, whereas metyrapone addition inhibited these activities by 20%. The study demonstrates several in vitro factors which alter AHH activity in primary cultures of rat mammary epithelial cells.

Induction of Casein Synthesis in a Rat Mammary Tumour Epithelial Cell Line by Dimethyl Sulphoxide

Induction of Casein Synthesis in a Rat Mammary Tumour Epithelial Cell Line by Dimethyl Sulphoxide