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.     

Growth of mouse mammary epithelium in response to serum-free media conditioned by mammary adipose tissue

Normal mouse mammary epithelial cells, isolated from female Balb/c mice, were cultured as multicellular organoids either on or within collagen gel matrices. Cultures were maintained in either serum-free control medium or the same medium conditioned by mammary adipose tissue. A significant proliferative response above that observed in control cultures (2.5-3.5 fold increase) was induced by conditioned medium derived from either mammary fat-pad explants or isolated adipocytes. In addition, scanning electron microscopy revealed epithelial morphology to be preserved in a more in vivo-like state in the conditioned medium. We conclude that diffusible factors derived from the mouse mammary fat pad influence the proliferative activity and morphology of mammary epithelial cells in culture.     

Identification of novel, stage-specific polypeptides associated with the differentiation of mammary epithelial stem cells to alveolar-like cells in culture

A linear pathway of morphologically intermediate cells has been identified between the cuboidal epithelial stem cells and the doming alveolar-like cultures of the cell line Rat Mammary (Rama) 25 in the order: cuboidal----grey----dark----dark droplet cell----doming cultures. The overall process can be accelerated by dimethyl sulfoxide (DMSO) or retinoic acid (RA) in the presence of mammotrophic hormones. From 400-450 [35S]methionine-labeled polypeptides that are routinely separated by two-dimensional gel electrophoresis approximately only 3% change during this process. As the Rama 25 cultures become confluent, three polypeptides of molecular weights (MW) 35 kD (pl = 7.7), 45 kD (pl = 7.5) and 33 kD (pl = 7.7) increase dramatically in radioactive abundance. These increases correspond to increases in numbers of grey cells for the 35 kD polypeptide, to increases in numbers of dark cells together with increases in peanut lectin-binding-ability for the 45 kD polypeptide, and to increases in the numbers of dark cells and in the numbers of droplet cells for the 33 kD polypeptide. After treatment with DMSO, RA or in spontaneously doming cultures, a second set of four polypeptides of MW 26 kD (pl = 5.9), 27 kD (pl = 6.2), 30 kD (pl = 7.2), and the same 33 kD polypeptide as above increase with the increase in numbers of droplet cells, domes, and increase in casein secretion. A variant of Rama 25, Rama 259, which fails to produce droplet cells, domes, or to secrete casein with DMSO and hormones also shows the same changes in the first set but not in the second set of polypeptides. The elongated, myoepithelial-like cell line derived from Rama 25, Rama 29, which cannot undergo any of the above intercellular conversions, fails to show changes in any of these polypeptides. Major changes in radioactive polypeptides have been confirmed for nonradioactive polypeptides and for polypeptides labeled for 4 hr with [35S]methionine. The synthesis of these novel polypeptides thus marks specific morphological stages of the differentiation of mammary epithelial stem to alveolar-like cells in culture, and as such may mark similar differentiation stages in vivo.     

Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial, and stromal cell lines in culture

A number of single-cell-cloned cell lines have been used to examine the growth-promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary-derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial-like cells. Epidermal growth factor (EGF) has a widespread but variable growth-stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 micrograms/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial-like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

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.     

Collagen-binding proteins of rat mammary tumor epithelial cells: a biochemical and immunological study

Collagen-binding proteins were studied in mammary epithelial cells of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. These proteins can be solubilized from cell membranes with 0.1% Triton. Using affinity chromatography on type I collagen-Sepharose and polyacrylamide slab gel electrophoresis, three major proteins of 34,000, 36,000, and 38,000 Da were found. Similar proteins were also present in several other cell types, including both epithelial and mesenchymal cells. Pulse-chase experiments did not indicate a precursor-product relationship of these proteins. Tryptic/chymotryptic peptide maps, however, revealed that the 36,000- and 38,000-Da proteins are very similar but are quite different from the 34,000-Da molecular form. The distribution and function of these proteins were then analyzed by using polyclonal antibodies directed against the entire set of major proteins. In immunofluorescence studies we observed a dense, punctate distribution of fluorescence on the cell surface of isolated and unfixed epithelial organoids and a bright pericellular staining in cultures after fixation. Treatment with the antiserum did not affect attachment and spreading of cuboidal mammary cells to plastic or to a collagen substratum. However, when the antiserum was added to the medium of growing cuboidal cells, it caused the formation of duct-like structures. These studies indicate that collagen-binding proteins may play a role in mammary gland morphology.     

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.     

A dual enzyme method for the establishment of long‐ and medium‐term primary cultures of epithelial and fibroblastic cells from Atlantic salmon gills

A dual enzyme disaggregation method using collagenase and then trypsin was developed that allowed the reproducible initiation of primary cultures from Atlantic salmon Salmo salar gills. Cultures had both epithelial and fibroblast morphology and persisted for an average of 20 passages. Growth was dependent upon a minimum concentration of 5% foetal calf serum (FCS) for fibroblasts and 10% FCS for epithelial cells. Growth was mostly independent of substrate, although epithelial cells showed increased growth on type I collagen gels. Matrigel? cell culture substrate produced reduced growth of fibroblasts and did not benefit epithelial cell growth. Epithelial cells reacted with monoclonal antibodies (MAbs) against mammalian cytokeratins, and fibroblast cells reacted with MAbs against mammalian fibronectin and type I collagen. The method also produced two long‐term cultures: one epithelial and one fibroblast that have been designated RGE‐2 and RGF respectively.     

Transplantation of a Mammary Stromal Cell Line into a Mammary Fat Pad: Development of the Site-Specific in Vivo Analysis System for Mammary Stromal Cells

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.     

Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.     

Human breast epithelium in vitro: The re-expression of structural and functional cellular differentiation in long-term culture

Summary Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/ cell contact or topological organisation of cells prior to embedding within the collagen matrix.     

Multifunctional phosphatidic acid signaling in mammary epithelial cells: Stimulation of phosphoinositide hydrolysis and conversion to diglyceride

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide-specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen-coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with ³H-myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E₂ affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with ³H-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride. © 1995 Wiley-Liss, Inc.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Secretory function of lactating mouse mammary epithelial cells cultured on collagen gels

Mammary epithelial cells dissociated from lactating mouse mammary glands form confluent monolayer cultures on collagen gel substrates. For these cultures, the substrate is more significant than the presence of lactogenic hormones in the maintenance of cell differentiation, as indicated by both morphological and biochemical criteria. Only cells cultured on floating collagen gels are able to maintain their lactose pool over several days in culture, although their ability to synthesize and secrete lactose becomes impaired. These cells are cuboidal in shape. In contrast, cells cultured on attached gels, which are constrained from changing shape and whose basolateral surfaces are inaccessible, lose their differentiation with time in culture. These flattened, dedifferentiated cells respond to the same hormonal environment by showing a mild proliferative response. Therefore, the response of cells to their hormonal milieu may be correlated with their shape: the squamous cells dedifferentiate and proliferate; the cuboidal cells maintain their differentiation and do not proliferate.     

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.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: Correlation with tumour type and progression

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.