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.     

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.     

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.     

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.     

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.     

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.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

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.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.     

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.     

Mammary gland morphogenesis in vitro: Formation of branched tubules in collagen gels by a cloned rat mammary cell line

The three-dimensional growth in vitro of cloned rat mammary cell lines on floating type I collagen gels has been investigated. Multicellular outgrowths formed by the various cell types show morphological differences on serial histological sectioning and electron microscopy. One cell line, Rama 25, an epithelial cell line derived from a dimethylbenz(a)anthracene (DMBA)-induced mammary adenocarcinoma can form branched tubules within the matrix. The amount of collagen in the matrix modified the structure of the predominant outgrowths formed by this cell line. High-concentration (0.6% w/v) collagen gels support the growth of tubules up to 0.5 mm in length which have an extensive lumen surrounded by rings of up to 26 cells. Absence of differentiated myoepithelial elements around the ring suggests a resemblence to primitive ducts found in the mammary glands of neonatal rats. The spectrum of cellular polarity toward the lumen seen throughout the tubules and the occasional irregular arrangement of epithelial cells are features of adenocarcinoma. Lumen formation occurs by central cell necrosis and separation of the external layers of initially solid cords. The tubules branch either dichotomously, by bifurcation at the distal ends or monopodially, by budding at the sides of the outgrowths. Rama 25 grown on gels containing lower concentrations of collagen (0.1 or 0.3% w/v) produce narrow branching structures with incomplete lumina and spikes of elongated cells. Tubular structures are not formed by Rama 25 grown on nonfloating gels. At the light microscopic level the layer of spindle cells formed beneath the surface monolayer on nonfloated gels resembles the sarcomatous regions of tumors, however at the ultrastructural level the spindle cells show some evidence of being myoepithelial-like rather than fibroblast-like. Sandwiching the epithelial cell sheet between two layers of collagen gel results in loss of contact with the media and the formation of spindle cells. The myoepithelial-like cell lines Rama 29 and Rama 401 form spiked branches of elongated cells and solid branching cords of cells, respectively. However, no lumen formation is observed. The fibroblast-like cell line Rama 27 shows extensive migration of either single cells or chains of cells into the gel. Thus only one cell type (Rama 25) is necessary to form branched tubules in vitro and the structure of the tubules can be modified by collagen, a component of the extracellular matrix.     

Role of fibronectin in the organisation of the cytoskeleton during the spreading of rat mammary epithelial cells.

The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor.     

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.     

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.     

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.     

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.     

Production of metastatic phenotype with DNA from metastatic cells but not with oncogenic DNA

Although the tumorigenic phenotype can be transferred by transfecting DNA from a variety of human tumours into 3T3 mouse fibroblasts, the ability to transfer the metastatic phenotype in a similar manner in syngeneic animals is largely unknown. We now report that transfection of a rat mammary epithelial cell line Rama 37, which produces nonmetastasizing adenomatous tumours in syngeneic rats, with DNA from a metastasizing rat mammary cell line, Rama 800, yields some transfectants with reproducible metastatic capabilities. Nonspecific DNA or the oncogenes EJ-ras-1 or Polyoma Large T Antigen fail in this respect, although their transfectants often possess increased tumorigenic potential.     

Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.