Sharon A. Ferns

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

Abstract 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.

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin.

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.

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

SummaryParenchymal 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.

Localization of vimentin in myoepithelial cells of the rat mammary gland

SummaryMyoepithelial cells in the virgin rat mammary gland have been shown to contain vimentin, using a polyclonal antiserum to vimentin purified from hamster fibroblasts. This antiserum has been shown to be specific for vimentin by immunoblotting and ELISA techniques. Similar results were obtained with a monoclonal antibody to vimentin. In the mammary glands of pregnant rats, the staining with vimentin antibodies is much weaker in the myoepithelial cells of the developing alveolar buds than in the main ducts. Similarly, in lactating glands, the staining of myoepithelial cells is much weaker in the secretory alveoli than in lactiferous sinuses. In each case, staining with antivimentin co-localizes with staining with polyclonal antisera to callous keratin (which specifically stain myoepithelial cells in the rat mammary gland).

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.

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.