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.     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

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.     

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.     

Bindings of the lectins Griffonia simplicifolia-1 and pokeweed mitogen mark discrete stages of myoepithelial-like differentiation of cell lines from the rat mammary gland

Individual single-cell-cloned cell lines of the different rat mammary (Rama) cell types have been tested for their ability to bind the lectins Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) using fluorescent, histochemical, and radioactive assays. Myoepithelial-like cell lines isolated from neonatal rat mammary glands and from nonmetastasizing tumors strongly bind GS-1 and PWM, whereas the corresponding epithelial and fibroblastic cell lines do not. When the epithelial cell lines are grown on floating gels of polymerised rat tail collagen, the basally situated or peripheral cells are stained strongly with peroxidase-conjugated lectins, whereas the apically or luminally situated cells are unstained. The capacity of cell lines intermediate in morphology between epithelial and myoepithelial-like cells to bind to GS-1 is as follows: Rama 25 epithelial less than Rama 25-12 less than Rama 25-11 less than Rama 25-14 less than Rama 29 myoepithelial-like cells, the same order as for other markers of myoepithelial cells. Conjugated PWM, however, binds only to the myoepithelial-like cell lines. Treatment of Rama 25 epithelial cells with agents that disrupt microtubules accelerates their conversion to elongated, myoepithelial-like cells in culture. The binding of cells to GS-1 is observed prior to, and that to PWM after, the major morphological change. It is suggested that the stepwise appearances of carbohydrate receptors for GS-1 and PWM mark discrete stages in the differentiation of epithelial to myoepithelial-like cells in culture, in the same way that they mark similar differentiation stages in ductal development in mammary glands of prepubertal rats.     

Proteins in cerebrospinal fluid and plasma of fetal rats during development

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.     

A possible mammary stem cell line

The cell line Rama 25 is derived from a mammary tumor induced in a rat by dimethylbenzanthracene. During rapid proliferation, Rama 25 cells appear as a single undifferentiated epithelial type; at high cell densities, however, small numbers of two other cell types are formed, which respectively resemble secretory and myoepithelial cells of the mammary gland, as judged by light and electron microscopy and immunofluorescent staining for casein (milk proteins). These additional cell types cannot be explained as contaminating cell populations since the cell line has been cloned several times; furthermore, the proportion of either can be increased by dimethylsulphoxide under different conditions. Specific epithelial features are seen by histological and ultrastructural examination of tumors formed by Rama 25 cells in immunodeficient mice. A line of the myoepithelial-like cells, Rama 29, isolated from a Rama 25 culture by cloning, is also described.We propose that the undifferentiated cell type is a form of mammary stem cell which can differentiate in culture.     

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.     

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.     

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.     

Isolation and properties of rat cell lines morphologically intermediate between cultured mammary epithelial and myoepithelial-like cells

The cloned cuboidal epithelial cell line Rat Mammary (Rama) 25 converts at low frequency in culture to elongated cells that possess some of the properties of myoepithelial cells; one such clonal cell line is termed Rama 29. Three morphologically intermediate clonal cell lines have been isolated from Rama 25 which form a morphological series in the order: Rama 25 cuboidal cells, Rama 25-Intermediate 2(I2), Rama 25-I1, Rama 25-I4, and Rama 29 elongated cells. This same order is largely maintained for increasing percentages of elongated cells, decreasing percentages of cuboidal cells, decreasing tubular structures on collagen gels, and increasing times of appearance of tumors in nude mice. The fully elongated cells fail to revert to cuboidal cells and to form tumors. Binding of antisera to epithelial-specific milk fat globule membranes and human keratin declines whereas binding of antisera to myoepithelial-associated laminin, vimentin, and Thy-1 increases in the cell lines in the same order. Similarly 7 polypeptides characteristic of elongated cells increase and 4 polypeptides characteristic of cuboidal cells decrease in the cell lines in the same way. Anti-actin serum binds equally to all cell lines grown on plastic, except for Rama 25-I4, where its binding is increased. Rama 25-I1 and Rama 25-I4 cells also give rise to anti-actin, anti-myoglobin, and phosphotungstic acid hematoxylin-staining giant, striated cells on collagen gels and in tumors that also have ultrastructural characteristics of skeletal muscle. Fresh elongated converts of Rama 25 bind appreciably more anti-actin serum than many of the clonal elongated cell lines such as Rama 29. Ultrastructural analysis confirms the gradual loss of epithelial characteristics and the acquisition of immature myoepithelial characteristics in the same sequence of cell lines. It is suggested that such a linear sequence of intermediate morphological states occurs between the Rama 25 cuboidal cells and the elongated myoepithelial-like cells in vitro, and that a similar morphological sequence may exist in terminal ductal structures in vivo.     

Redistribution of fibronectin and cytoskeletal proteins during the differentiation of rat mammary tumor cells in vitro

The tumorigenic mammary epithelial stem cell line, Rama 25, is capable of synthesizing and secreting fibronectin but incorporates only small amounts of fibronectin into pericellular material localised in regions of cell-cell and cell-substratum contact. Under certain culture conditions, Rama 25 differentiates into a non-tumorigenic myoepithelial-like cell line, Rama 29, which is capable of retaining fibronectin on the cell surface in characteristic fibrillar formation. The redistribution of fibronectin is accompanied by a reorientation of the cytoskeleton from circular bundles in Rama 25 to parallel arrays of filaments in Rama 29. In vivo, fibronectin is found in the basement membrane of the mammary gland and our in vitro studies lead us to suggest that the mammary myoepithelial cell in vivo synthesizes much of the basement membrane fibronectin.     

Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland

A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes project from the cell surface and numerous membrane pinocytotic vesicles can be seen. The cytoplasm is filled with linear arrays of 5- to 7-nm filaments with occasional dense foci. Cell junctions with associated 8- to 11-nm tonofilaments are also observed. Immunofluorescence techniques reveal actin and myosin filaments and also intermediate filaments of both prekeratin and vimentin types. Rama 401 cells secrete an amorphous material that, when an immunoperoxidase technique is used, stains with antibodies to basement membrane-specific type IV collagen. Localized densities of the cell membrane, which resemble hemidesmosomes, are located adjacent to these extracellular deposits. Immunofluorescence staining and immunoprecipitation techniques reveal that the cells also synthesize two other basement membrane proteins, laminin and fibronectin. The type IV collagen consists of two chains with molecular weights of 195,000 and 185,000.     

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties

Summary EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H₁B clone form branching cords devoid of a central lumen, K₃A₃ cells from cords enclosing small multifocal lumina, and J₃B₁ cells form large cavitary structures containing a wide lumen. I₃G₂ cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Be1a cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell-lined cavitary structures, except H₁B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesis.     

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.     

Similar growth pattern of mouse mammary epithelium cultivated in collagen matrix in vivo and in vitro

Mouse mammary ductal cells cultured in type I collagen gels give rise to three-dimensional multicellular outgrowths consisting of thin spikes which are often branched, and which may have pointed or blunt ends. The significance of these spikes to normal ductal morphogenesis has been unclear, since identical structures are not known to occur in vivo; conversely, it has not been possible to maintain in gel culture the highly structured end buds which are characteristic of ductal elongation in the animal. In order to evaluate whether the pattern of radiating spikes observed in collagen gel cultures results from chemical or physical peculiarities of the culture environment, a small volume of unpolymerized type I collagen solution was injected into mammary gland-free fat pads of young adult mice. After the bubble of collagen had polymerized, an implant of mammary ductal epithelium was introduced into the center of the gel. Histological examination of the implants after 3 to 6 days of growth revealed numerous small epithelial spikes, similar to those observed in gel culture, extending into the fibrous matrix. The early stages of regeneration of mammary implants placed in gland-free fat pads were then examined without the addition of exogenous collagen. In cases where the epithelium happened to contact a fibrous region of the fatty stroma, spikes were also seen to form in these natural collagenous substrates. Whether or not exogenous collagen was used, normal end buds were formed only when epithelial spikes contacted adipocytes. It was concluded that the three-dimensional pattern of radiating tubules in collagen gels in vitro is not merely an artifact of culture, but has a counterpart in vivo whereever regenerating mammary epithelium is surrounded by fibrous stroma. A model is presented in which the pattern of epithelial outgrowth is determined by the physical characteristics of the surrounding stroma; in collagen matrix a comparatively primitive and unspecialized type of morphogenesis occurs which may not require the participation of stromal cells. In contrast, epithelial-adipocyte interactions appear to be necessary for the formation of end buds and subsequent morphogenesis of fully structured mammary ducts.     

Epithelial organoids and mononuclear phagocytes from DMBA-induced mammary tumors of the rat secrete collagenase in vitro

The production of collagenase has been examined in primary cultures of multicellular epithelial organoids and of stromal cells isolated from DMBA-induced mammary tumors of the rat. Plastic culture dishes and dishes coated with collagen fibrils were used to study the effect of such a substrate on collagenase release. Cultures of 51-μm epithelial organoids consisted of cuboidal cells and a myoepithelial-like cell type which formed a continuous layer under the cuboidal cells. A transient low production of collagenase with an apparent molecular weight (MW) of 72 kD was detected on both substrates. Upon separation by trypsin only cuboidal cells released collagenase. Cultures of 27-μm organoids contained only few myoepithelial-like cells. On plastic, they formed dense monolayers of cuboidal cells and released more collagenase than the greater aggregates. On collagen fibrils, these organoids formed cords and ridges and collagenase production was about 4- to 6-fold higher. These results indicate that collagenase release is influenced by the nature of the interaction of cuboidal cells with the substrate on which they grow. Similar organoids prepared from virgin mammary glands failed to secrete collagenase on either substrate. Primary cultures of stromal cells derived from tumor tissues comprised one basic cell type that expressed a series of properties characteristic for monocytes/macrophages. These cultures were capable of producing collagenase with an apparent MW of 56 kD. Collagenase with a similar size was detected in the extracts of 51 from 65 mammary tumors.     

Chromogranin A alters ductal morphogenesis and increases deposition of basement membrane components by mammary epithelial cells in vitro.

The extracellular function of chromogranin A (CgA), a glycoprotein widely distributed in secretory vesicles of neurons and neuroendocrine cells, has not been clearly established. To examine whether CgA might modulate the biological properties of epithelial cells, we used an in vitro model of ductal morphogenesis in which mammary epithelial (TAC-2) cells are grown in three-dimensional collagen gels. Whereas under control conditions TAC-2 cells formed thin, branched cords with pointed ends, in the presence of CgA they formed thicker cords with bulbous extremities, reminiscent of growing mammary ducts in vivo. Immunofluorescence analysis demonstrated that CgA increases the deposition of three major basement membrane components, i.e., collagen type IV, laminin, and perlecan, around the surface of the duct-like structures. Similar effects were observed with CgA partially digested with endoproteinase Lys-C, suggesting that one or more fragments of CgA are endowed with the same activity. These findings reveal a hitherto unsuspected activity for CgA, i.e., the ability to alter ductal morphogenesis and to promote basement membrane deposition in mammary epithelial cells.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.