Control of type IV collagen production in rat mammary epithelial and myoepithelial-like cells

A rat mammary myoepithelial-like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative "dot" hybridization techniques, the level of poly (A)-containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5-fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 71% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.     

Isolation of simian virus 40-transformed human mammary epithelial stem cell lines that can differentiate to myoepithelial-like cells in culture and in vivo

Simian Virus 40 (SV40) transformation of primary cultures of human mammary epithelial cells has yielded a cloned epithelial-like cell line and a representative, single-cell subclone. Although apparently homogeneous, both cloned cell lines can also yield small numbers of three other cell types. The more-elongated cell type can be obtained directly by replating cells from the medium of the epithelial-like cell cultures or by picking and culturing single cells to form representative lines. Immunofluorescent and immunocytochemical analysis of these cell lines growing on plastic or as tumor-nodules in nude mice for epithelial membrane antigens, various cytokeratins, various actins, laminin, Type IV collagen, the common acute lymphoblastic leukemia antigen (CALLA), and a 135-kDa glycoprotein confirm the epithelial nature of the epithelial-like cells and suggest a myoepithelial origin for the more-elongated cell type. Ultrastructural analysis largely confirms the results, although the myofilamental bundles can be scanty in the growing myoepithelial-like cells. The other two cell types are possibly related to the keratinizing and casein-secreting cells seen in the epithelial tumor-nodules before and after mating the mice, respectively. The myoepithelial-like cells produce 5- to 17-fold more laminin, Type IV collagen, CALLA, and the 135-kDa glycoprotein than the epithelial cells, and all of these antigens are preferentially found on myoepithelial cells in vivo. It is suggested that the SV40-transformed epithelial cell is an immortalized form of human mammary stem cell which can differentiate in culture and in vivo to myoepithelial-like cells.     

Isolation and differentation of cloned epithelial cell lines from normal rat mammary glands

Summary Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels with form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cel lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pincocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.     

Extracellular matrix control of collagen production by rat mammary epithelial and myoepithelial-like cells in vitro

A rat mammary myoepithelial cell line (Rama 401) grown on plastic produces 5 times more collagen (largely type IV) than a mammary epithelial cell line (Rama 704) grown on the same surface. When the cells are grown on collagen gels, the amount of collagen produced by Rama 704 cells increases 3.3 times, whereas there is no increase in collagen production by Rama 401 cells. Increased production of collagen by Rama 704 cells is due to both an increased rate of synthesis and a decreased rate of degradation. These results indicate that for mammary epithelial cells, unlike myoepithelial cells, the rate of production of collagen can be regulated by the extracellular matrix.     

Elongated cells derived from rat mammary cuboidal epithelial cell lines resemble cultured mesenchymal cells in their pattern of protein synthesis

Two-dimensional gel electrophoresis has been used to identify polypeptide patterns characteristic of rat mammary cuboidal epithelial cells or mesenchyme-derived cells. Elongated cells and cell lines derived from cloned cuboidal epithelial cells in culture possess a polypeptide pattern which resembles that of the cultured mesenchymal cells rather than that of the cuboidal epithelial cells from which they were derived. These elongated converts also resemble cultured mesenchymal cells in possessing a Triton-insoluble matrix in which vimentin and not prekeratin predominates.     

Synthesis of basic fibroblast growth factor upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.     

Isolation of rat mammary epithelial cells for polyamine analysis.

Mammary epithelial cells were isolated from either abdominal-inguinal glands or mammary tumours of rats, after enzymic digestion of the tissues, and were analysed for polyamine content. Optimum conditions were developed for the isolation of cells in sufficient yield for the analysis of polyamines from 1 g of mammary gland or 0.5 g of tumour tissue. Complete recoveries of the polyamines in the tissues were achieved in the isolated epithelial cells.     

Differentiation of simian virus 40 transformed human mammary epithelial stem cell lines to myoepithelial-like cells is associated with increased expression of viral large T antigen

Cloned simian virus 40 (SV40)-transformed human breast epithelial cell lines can differentiate to myoepithelial-like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor-nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial-like cells and cell lines. The myoepithelial-like cell lines produce 4-6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5-5-fold in the myoepithelial-like cell lines when analysed by dot-blot or by Northern hybridisations. Thus, differentiation along the myoepithelial-like cell pathway is associated in these SV40-transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.     

Increased abundance of a normal cell mRNA sequence accompanies the conversion of rat mammary cuboidal epithelial cells to elongated myoepithelial-like cells in culture.

Rat mammary cuboidal epithelial cell lines in culture convert to elongated myoepithelial-like cells. This conversion is accompanied by the appearance of a 9,000 molecular weight acidic polypeptide (p9ka), abundant in the elongated convertants, but which is hardly detectable in the cuboidal epithelial cells. A cDNA library corresponding to a low-molecular-weight fraction of poly(A)- containing RNA from a myoepithelial-like cell line, has been constructed. Recombinant plasmids containing cDNA complementary to p9ka mRNA have been identified by hybrid-selected translation. The mRNA for p9ka has been identified by Northern blotting and is found to be at least five-times more abundant in cultured myoepithelial-like rat mammary cells when compared to the cuboidal epithelial cells. This cytoplasmic mRNA sequence, which is present in increased abundance in cultured mammary myoepithelial-like cells, is also present, at lower levels, in normal rat tissues, including the mammary glands.     

Microtubule-disrupting drugs increase the frequency of conversion of a rat mammary epithelial stem cell line to elongated, myoepithelial-like cells in culture

Rat mammary (Rama) 25 cuboidal epithelial stem cells convert at a low frequency to elongated, Thy-1-positive, myoepithelial-like cells in culture; one such cell line is termed Rama 29. Addition of increasing concentrations of the microtubule-disrupting drug colchicine to sparse cultures of Rama 25 dramatically increases the percentage of colonies containing elongated cells and the percentage of Thy-1-positive cells when the drug is removed. Similar results on the formation of elongated cell colonies are obtained with other microtubule disruptors, such as vinblastine, vincristine, demecolcine, and nocodazole. The inactive analogues of colchicine beta- and delta-lumicolchicine and the microfilamental-disruptors cytochalasin B and D are without effect on the formation of elongated cell colonies and Thy-1-positive cells. For a given concentration of colchicine the percentage of elongated cell colonies and Thy-1-positive cells increases the longer the cells are exposed to the drug (range 8-96 hr) and the longer the drug-treated cultures are subsequently grown in drug-free medium. Colchicine fails to display this morphological change on Rama 29 elongated cells and on Rama 600 epithelial cells from a rat mammary metastasizing tumor. Immunofluorescent localization of antisera to tubulin confirms that colchicine disrupts the microtubules in all three cell lines at similar concentrations (0.1 to 1 microM) to those required to increase the percentage of elongated cell colonies in Rama 25. The DNA synthesis inhibitor cytosine arabinoside fails to inhibit this conversion process, and time-lapse cinematographic studies confirm that the conversion of a cuboidal to an elongated cell can take place without cell division. However, cell division may sometimes be required for subsequent stabilization events. Treatment of Rama 25 cells with colchicine under the same conditions also increases the abundance of elongated cell (Rama 29)-associated polypeptides, and elongated cell clones isolated after such treatment show an overall pattern of protein synthesis very similar to that of Rama 29.     

Appearance of basic fibroblast growth factor receptors upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

The binding of [125I]-epidermal growth factor (EGF) and [125I]-basic fibroblast growth factor (bFGF) to a number of single-cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high-affinity [125I]-EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial-like cell lines. In contrast, high-affinity (KD 35-276 pM) [125I]-bFGF binding sites were present on fibroblastic and myoepithelial-like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial-like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity-cross-linked to [125I]-bFGF were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.     

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.     

Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

Establishment and characterization of bovine mammary epithelial cell lines

Summary One bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithelial cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secretingα-lactalbumin andα-s₁-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation. This work was supported by the Pennsylvania State University Experiment Station. The PS-BME cells are the property of The Pennsylvania Research Corporation. Scientists interested in obtaining the PS-BME clone or cell lines for their research may request them from the corresponding author.     

Human mammary cancer cell lines and other epithelial cells cultured as organoid tissue in lenticular pouches of reinforced collagen membranes

Summary A system has been developed for the culture of cells that provides conditions favoring the formation of tissues comparable to conditions existing in nature. The culture chamber is a lens-shaped pouch composed of two thin-walled, reinforced, waffled collagen membranes facing each other. The chamber is immersed in immersed in medium in a closed transparent container and incubated on a rocker. On histologic study, after days to weeks in culture, human mammary cancer cell lines BT-20, MCF-7, MDA-231, MDA-468, and T47D grow in the chamber as distinctive structured epithelial tissue. Dog kidney cell line MDCK grows as a papillary adenocarcinoma and rat bladder cancer line NBT-II as an epidermoid carcinoma; cells from clinical effusion tumors produce distinct tissue. Changes in histologic phenotype may be driven by molecular changes at the level of the genome. Resulting alteration of the biochemical functions essential for the integrity of specific durable tissue organization should alter or reset the pattern of tissue organization and of biological behavior, including malignancy and response to cytotoxic chemicals. Lenticular pouch culture promises to be an effective tool for exploring the molecular changes associated with histogenesis and malignancy. This paper is dedicated to the memory of Clyde J. Dawe, who died suddenly on Cape Cod, Massachusetts, in a glider accident on July 5, 1996. We were friends for about 40 years. Clyde was one of the finest scholars in cancer biology I have ever known. I learned many things from him and miss him.     

Comparative properties of untreated andN-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines

Summary A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eightN-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngenic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.     

Isolation of two morphologically distinct cell lines from rat arterial smooth muscle expressing high tumorigenic potentials

Summary Smooth muscle cell proliferation is an important feature of atherogenesis. Some works have hypothesized that a transformation of smooth muscle cells could arise during this pathological process. The present paper describes two spontaneously transformed cell lines of arterial smooth muscle cells (SMC) established from aortic media of adult rat. The cell lines have been designated V6 and V8; some of their morphologic, growth, and metabolic characteristics are described and compared to their parent cells. The two cell lines appeared distinct by their morphology and by their degree of transformation. V6 cells appeared as elongated spindle-shaped cells whereas V8 cells were spread cells with a cobblestone pattern. Karyotypes of both cell lines showed a high polyploidy level. V6 and V8 cell lines were immortalized and showed growth characteristics of transformed cells: low requirement of serum to grow, ability to form colonies in soft agar and tumorigenicity in nude mice; V8 cells presented a higher malignancy than V6 cells. Both V6 and V8 cells exhibited characteristics of cultured arterial SMC: ultrastructure, alpha actin expression at the protein and mRNA level, prostacyclin production. The remarkably different morphologies of the V6 and V8 lines and their transformed phenotype suggest that these cell lines could be useful models to study SMC differentiation and proliferation with respect to atherosclerotic or hypertensive vascular diseases. Electron microscopy was performed in the Center of Electron Microscopy Applied to Biology and Geology (CEMABG), Claude Bernard University, Lyon I. Flow cytofluorometry was performed in the Center of Fluorometry, Department of Human Biology, Claude Bernard University, Lyon I and funded by ARC N^o 6055-80. This work was supported by INSERM, by MRT grant 86-C-0301 and by ARC grant 415-87.