共查询到20条相似文献,搜索用时 15 毫秒
1.
M J Warburton S A Ferns N E Hynes 《Biochemical and biophysical research communications》1986,137(1):161-166
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. 相似文献
2.
Phosphoinositide-3 kinase-Rac1-c-Jun NH2-terminal kinase signaling mediates collagen I-induced cell scattering and up-regulation of N-cadherin expression in mouse mammary epithelial cells 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Molecular biology of the cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I-induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I-induced N-cadherin up-regulation. These data implicate PI3K-Rac1-JNK signaling in collagen I-induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I-induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I-induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K-Rac1-JNK signaling. 相似文献
3.
4.
Muthulekha Swamydas Jill M. Eddy Karen J. L. Burg Didier Dréau 《In vitro cellular & developmental biology. Animal》2010,46(8):673-684
Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here, we present the effects of various 3D in vitro stroma compositions (termed “matrices” or “substrates”) on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures. Normal murine mammary gland (NMuMG) cells were cultured, either alone or in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices generated by agarose, collagen, and Matrigel® alone or by a combination thereof. After 3–5 d in culture, cell distribution, organization, and the presence of acinus-like and tubule-like structures were determined. The number of acinar structures was significantly higher in cultures grown in combination matrices of agarose with Matrigel® or collagen I when compared with cultures grown in Matrigel® or collagen I alone (p?0.05). No tubular structures were formed when agarose was included in the matrix, regardless of the combination. In Matrigel®, but not in collagen I/Matrigel® microenvironment, the number of tubular structures was significantly increased in NMuMG/D1 coculture when compared with culture of NMuMG cells alone (p?0.05). By immunohistochemical analysis, NMuMG cells cocultured with D1 cells were shown to form acinar structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral. Immunostaining for laminin indicated the presence of basement membrane when NMuMG cells were grown in Matrigel® alone or cocultured with D1 cells in a combination of Matrigel® and collagen I. These results indicate that the physical and biochemical properties of the matrix and cellular composition alter the organization of the mammary gland. 相似文献
5.
Cripto-1 enhances migration and branching morphogenesis of mouse mammary epithelial cells 总被引:3,自引:0,他引:3
Wechselberger C Ebert AD Bianco C Khan NI Sun Y Wallace-Jones B Montesano R Salomon DS 《Experimental cell research》2001,266(1):95-105
Cripto-1 is an EGF-CFC protein that performs an important role during early vertebrate development and is overexpressed in several types of human cancer. In the present study mouse EpH4, NMuMG, and TAC-2 mammary epithelial cells that are negative for endogenous cripto-1 expression were transfected with the murine cripto-1 cDNA. Cripto-1-transfected cell lines exhibited functional and physiological differences from the original cell lines including enhanced anchorage-independent growth in soft agar (EpH4 cells), growth in serum-free medium, increased proliferation, and formation of branching, duct-like structures when grown in a three-dimensional collagen type I matrix. Furthermore, cripto-1-expressing cell lines showed elevated migration in vitro in Boyden chamber and wound-healing assays. These results indicate that cripto-1 can function through an autocrine pathway that enables mammary epithelial cells to undergo an epithelial to mesenchymal transition. 相似文献
6.
Desrivières S Prinz T Castro-Palomino Laria N Meyer M Boehm G Bauer U Schäfer J Neumann T Shemanko C Groner B 《Molecular & cellular proteomics : MCP》2003,2(10):1039-1054
Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process. 相似文献
7.
Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens 总被引:32,自引:0,他引:32
A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treatment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete proteoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, III, and V but not types II, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminoglycans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell surface heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices. 相似文献
8.
TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors 总被引:15,自引:0,他引:15
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1994,127(6):2021-2036
The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E- cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta- mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition. 相似文献
9.
Heparan sulfate proteoglycans from mouse mammary epithelial cells: localization on the cell surface with a monoclonal antibody 总被引:28,自引:13,他引:15
Mouse mammary epithelial cells, of the normal murine mammary gland (NMuMG) cell line, bear a heparan sulfate-rich proteoglycan (HSPG) on their surfaces. A hybridoma (281-2) secreting a monoclonal antibody that recognizes this HSPG was produced by fusion of SP-2/0 myeloma cells with spleen cells from rats immunized with NMuMG cells. The 281-2 monoclonal antibody is directed against the core protein of the cell surface HSPG, as demonstrated by (a) recognition of the isolated proteoglycan but not its glycosaminoglycan chains, (b) co-localization of 281-2-specific antigen and radioactive cell surface HSPG on gradient polyacrylamide gel electrophoresis and on isopycnic centrifugation, and (c) abolition of immunofluorescent staining of the NMuMG cell surface by the intact, but not the protease-digested ectodomain of the cell surface HSPG. The antibody is specific for cell surface HSPG and does not recognize the HSPG that accumulates extracellularly beneath the basal cell surface. Therefore, the 281-2 antibody may be used to isolate the cell surface HSPG and to explore its distribution in tissues. 相似文献
10.
Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins 总被引:1,自引:0,他引:1
Daniel S. Liscia Amelia Bernardi Bona Griselli Alberto P. M. Cappa 《In vitro cellular & developmental biology. Plant》1988,24(3):183-187
Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins
and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels
failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore
cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass
or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen
these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular
matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that
collagen substrates permit epithelial cell cultures to progress toward a more differentiated state.
Supported by grants from the Italian Assciation for Cancer Research (AIRC). 相似文献
11.
Shahnaz Hashmi Dairkee Carlene M. Blayney David M. Asarnow Helene S. Smith Adeline J. Hackett 《In vitro cellular & developmental biology. Plant》1985,21(6):321-327
Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to
vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary
cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody,
whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and
in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture.
Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations
of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin
found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly
as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the
tissue of origin 相似文献
12.
13.
Kondo M Cubillo E Tobiume K Shirakihara T Fukuda N Suzuki H Shimizu K Takehara K Cano A Saitoh M Miyazono K 《Cell death and differentiation》2004,11(10):1092-1101
Epithelial-mesenchymal transdifferentiation (EMT) is a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors. EMT can be induced by transforming growth factor (TGF)-beta in mouse NMuMG mammary epithelial cells. Here, we demonstrate a central role of helix-loop-helix factors, E2A and inhibitor of differentiation (Id) proteins, in TGF-beta-induced EMT. Epithelial cells ectopically expressing E2A adopt a fibroblastic phenotype and acquire migratory/invasive properties, concomitant with the suppression of E-cadherin expression. Id proteins interacted with E2A proteins and antagonized E2A-dependent suppression of the E-cadherin promoter. Levels of Id proteins were dramatically decreased by TGF-beta. Moreover, NMuMG cells overexpressed Id2 showed partial resistance to TGF-beta-induced EMT. Id proteins thus inhibit the action of E2A proteins on the expression of E-cadherin, but after TGF-beta stimulation, E2A proteins are present in molar excess of the Id proteins, thus over-riding their inhibitory function and leading to EMT. 相似文献
14.
New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line. 总被引:6,自引:3,他引:3
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
N E Hynes R Jaggi S C Kozma R Ball D Muellener N T Wetherall B W Davis B Groner 《Molecular and cellular biology》1985,5(1):268-272
A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes. 相似文献
15.
16.
Erika Schmid Werner W. Franke Christine Grund Dorothea L. Schiller Heiderose Kolb Neidhard Paweletz 《Experimental cell research》1983,146(2):309-328
Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed. 相似文献
17.
Mazzoni E Adam A Bal de Kier Joffe E Aguirre-Ghiso JA 《Molecular cancer research : MCR》2003,1(10):776-787
We have investigated the role of a classical isoform of protein kinase C (PKCgamma) in promoting immortalized mammary cell tumorigenesis in vivo and the contribution of proteases and adhesion molecules to this process. We hypothesized that overexpression of PKCgamma in immortalized mammary epithelial cells may initiate, by activating the mitogenic ERK pathway, early changes in proteases, adhesion molecules, and markers of an epithelium-to-mesenchyme transition that may contribute to in vivo tumorigenesis. Here we show that compared to vector-transfected cells, immortalized murine mammary epithelial cells (NMuMG) overexpressing PKCgamma have stronger activation of (approximately 5-fold) ERK1/2 MAPKs, which results in a similar increase in cyclin D1. In addition, PKCgamma-expressing cells showed increased levels of vimentin, fibronectin (FN), beta1-integrins, enhanced adhesion to fibronectin, and its organization into fibrils. Concomitantly, PKCgamma induced a dramatic down-regulation of E-cadherin protein levels and its localization to cell-cell junctions. NMuMG cells expressing PKCgamma became resistant to death by anoikis and formed colonies in soft agar. This effect was dependent on ERK activation, because Mek1/2 inhibition with PD98059 abrogated anchorage-independent growth. Most importantly, unlike control NMuMG cells, PKCgamma-transfected cells inoculated s.c. into nude mice displayed tumorigenic and invasive capacity and were able to spontaneously metastasize. This behavior correlated with increased production of uPA and MMPs-9/-2 induced by PKCgamma. These results suggest that PKCgamma overexpression in immortalized mammary epithelial cells may generate, through an increase in ERK, signaling changes in the expression of genes associated with an epithelium-to-mesenchyme transition that may be sufficient to favor tumor growth in vivo. 相似文献
18.
Chen H Pimienta G Gu Y Sun X Hu J Kim MS Chaerkady R Gucek M Cole RN Sukumar S Pandey A 《Proteomics》2010,10(21):3800-3810
The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients. 相似文献
19.
Establishment and characterization of a caprine mammary epithelial cell line (CMEC) 总被引:12,自引:0,他引:12
Pantschenko AG Woodcock-Mitchell J Bushmich SL Yang TJ 《In vitro cellular & developmental biology. Animal》2000,36(1):26-37
Summary We describe the establishment of a continuous, nontransformed cell line obtained from primary culture of a lactating (114
days postparturition) Anglo-Nubian (Capra hircus) goat mammary gland biopsy. These cells (CMEC), have been cultured in the presence of supraphysiologic concentrations of
insulin and hydrocortisone for more than 560 population doublings (over 80 passages) without any sign of senescence while
maintaining a normal/near-normal diploid chromosome modal number of 2n=60 and are responsive to contact inhibition of proliferation.
Cytoskeletal analysis indicates that CMECs are epithelial, without detectable fibroblastic or myoepithelial cells. When grown
at low density on plastic substratum, the cells tend to form island monolayer aggregates with the characteristics cobblestone
morphology of epithelial cells. With increasing density, the cells organize into lumen-like structures with various morphology
consisting of large and small vacuolized and nonvacuolized cells. Postconfluent cultures form epithelial raised dome-like
structures, implying a process of contact-induced differentiation. This is corroborated by positive immunocytochemistry to
lactation-specific proteins: β-casein and α-lactalbumin, which were predominantly expressed in dome-forming cells. We also
observed an overall modulation of cytokeratin 18/19 expression associated with number of days post subculture and with the
expression of lactation-specific proteins. Postconfluent cultures which contain lactation-specific, antibody-reactive, dome-like
structures showed a decreased expression of keratin 18 and no (null) expression for keratin 19. Lastly, cells cultured within
a collagen matrix show morphological differentiation with the organization of branching duct-like and acini-like structures.
This study suggests that CMECs are a useful in vitro model for study of mammary gland development and differentiation, in
particular, direct modulation of epithelial cells grown on plastic substratum or extracellular matrix without the influence
of stromal elements or the necessity and variability associated with primary cell culture or tissue explants. 相似文献
20.
Collagen-binding proteins were studied in mammary epithelial cells of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. These proteins can be solubilized from cell membranes with 0.1% Triton. Using affinity chromatography on type I collagen-Sepharose and polyacrylamide slab gel electrophoresis, three major proteins of 34,000, 36,000, and 38,000 Da were found. Similar proteins were also present in several other cell types, including both epithelial and mesenchymal cells. Pulse-chase experiments did not indicate a precursor-product relationship of these proteins. Tryptic/chymotryptic peptide maps, however, revealed that the 36,000- and 38,000-Da proteins are very similar but are quite different from the 34,000-Da molecular form. The distribution and function of these proteins were then analyzed by using polyclonal antibodies directed against the entire set of major proteins. In immunofluorescence studies we observed a dense, punctate distribution of fluorescence on the cell surface of isolated and unfixed epithelial organoids and a bright pericellular staining in cultures after fixation. Treatment with the antiserum did not affect attachment and spreading of cuboidal mammary cells to plastic or to a collagen substratum. However, when the antiserum was added to the medium of growing cuboidal cells, it caused the formation of duct-like structures. These studies indicate that collagen-binding proteins may play a role in mammary gland morphology. 相似文献