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1.
The potential calcium-binding protein p9Ka is related to S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. p9Ka accumulates abundantly in cultured rat mammary myoepithelial-like cells but is very much less abundant in the parental cuboidal epithelial cells. p9Ka mRNA is found in normal rat mammary gland, and preliminary experiments suggest that it is found in the mammary myoepithelial cells. A 17-kilobase pair fragment of cloned normal rat DNA contains the gene for p9Ka, but it also contains the gene for two additional polypeptides of molecular mass 6 kDa that are resolved as two isoelectric focusing variants by two-dimensional gel electrophoresis. These two isoelectric focusing variants correspond to two abundant polypeptides present in the cultured myoepithelial cells and probably arise from postsynthetic modification of the product of a single gene. The mRNA for the product of this gene and the p9Ka mRNA are both found in the normal rat mammary gland, but these two mRNAs are differentially expressed in certain tumor-derived rat cell lines.  相似文献   

2.
3.
Calcium-ion binding by the potential calcium-ion-binding protein, p9Ka   总被引:1,自引:0,他引:1  
p9Ka is a polypeptide of apparent molecular mass 9 kDa, present in cultured rat mammary myoepithelial-like cells, but virtually absent in their parental epithelial cells. mRNA for p9Ka is present in normal rodent tissues. The amino acid sequence of a protein of molecular mass 12 KDa, derived from the nucleotide sequence of the p9Ka gene, is related to that of S-100 protein, a calcium-ion-binding protein. p9Ka, isolated from cultured rat mammary myoepithelial-like cells is now shown to bind calcium ions in vitro suggesting that the derived amino acid sequence is correct, and that an apparent discrepancy between the molecular masses of the predicted and isolated p9Ka does not affect this activity.  相似文献   

4.
Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.  相似文献   

5.
Two-dimensional gel electrophoresis has been used to identify polypeptide patterns characteristic of rat mammary cuboidal epithelial cells or mesenchyme-derived cells. Elongated cells and cell lines derived from cloned cuboidal epithelial cells in culture possess a polypeptide pattern which resembles that of the cultured mesenchymal cells rather than that of the cuboidal epithelial cells from which they were derived. These elongated converts also resemble cultured mesenchymal cells in possessing a Triton-insoluble matrix in which vimentin and not prekeratin predominates.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
The binding of [125I]-epidermal growth factor (EGF) and [125I]-basic fibroblast growth factor (bFGF) to a number of single-cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high-affinity [125I]-EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial-like cell lines. In contrast, high-affinity (KD 35-276 pM) [125I]-bFGF binding sites were present on fibroblastic and myoepithelial-like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial-like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity-cross-linked to [125I]-bFGF were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.  相似文献   

16.
17.
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.  相似文献   

18.
The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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