Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of beta-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of beta-casein MRNA. The inducibility of beta-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, fibronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of beta-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.     

Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.     

Selenium retention and inhibition of cell growth in mouse mammary epithelial cell lines in vitro

The steady state levels of growth inhibitory doses of inorganic selenium were examined in five different mammary epithelial cell lines: MOD, COMMA-D, COMMA-F, COMMA-T, and YN-4. The retention of selenium was monitored using a radioactive isotope,⁷⁵Se. Growth inhibition correlated with high levels of selenium in the cell. Generally, the retention of intracellular selenium was not dependent upon cell density, cell number, net growth rate, or tumorigenicity of the mammary cell lines. One cell line, COMMA-D, exhibited an unique response wherein the amount of selenium retained was low and the growth inhibitory effects of selenium were negligible when the cells were exposed to selenium at low density. However, at high cell densities, the COMMA-D cells responded like the other four cell lines. The growth inhibitory effect of selenium was reversible; upon removal of selenium from the medium, cells start synthesizing DNA within 24h. The retention of selenium was influenced by constituents in the growth medium. In particular, cysteine, but not methionine, purines, or pyrimidines altered selenium retention and counteracted the growth inhibitory effects of selenium. These results indicated that the mammary cell lines, particulary COMMA-D and MOD are good model systems to examine the uptake, retention, localization, and function of inorganic selenium under conditions where it acts as a growth inhibitory agent.     

Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas.

The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic-type tissue in the adult rat liver can be experimentally induced (Reddy et al.: J. Cell Biol., 98:2082-2090, 1984; Rao et al., J. Histochem. Cytochem., 34:197-201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light- and electron-microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and beta-tubulin were present in all cell lines. In addition, simple epithelial type II cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type II cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth-inhibitory effects of TGF-beta 1. No expression of tissue- or cell-specific proteins such as alpha-fetoprotein, albumin, amylase, elastase, or gamma-glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a "precursor" or "stem" cell compartment in vivo.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Cellular composition and organization of ductal buds in developing rat mammary glands: evidence for morphological intermediates between epithelial and myoepithelial cells

In the developing rat mammary gland, terminal end buds (TEBs), lateral buds and alveolar buds represent the major sites of morphogenetic activity and cellular differentiation. The morphology and cellular composition of these buds from 20-to 22-day-old rats and cycling rats have been studied by immunocytochemical and electron microscopic techniques. The mammary buds are composed of a heterogeneous collection of cells including epithelial and myoepithelial cells, irregular loosely adherent cells, and occasional large clear cells. The irregular, loosely packed cells or cap cells are mainly situated around the periphery of the TEBs and lateral buds. "Chains" of irregularly shaped cells also extend from the peripheral cap cell layer to the center of the TEB; and, where they converge on lumina, they display microvilli and junctional complexes. At the tips of the end buds, the cap cells are of undifferentiated appearance; however, similar cells situated toward the subtending mammary ducts show a gradation in ultrastructure to that of myoepithelial cells. This change is accompanied by an increase in the amounts of immunoreactive myosin and keratin seen within the cells and a 200-fold increase in the thickness of the basement membrane. In contrast, the peripheral cells of the alveolar buds are more closely packed, contain a greater number of myofilaments, and show increased staining with antisera to myosin. We suggest that the undifferentiated cap cells do not represent a discrete cell type, since they show transitional forms to myoepithelial cells within the subtending mammary ducts, and that the tendency toward the myoepithelial phenotype is predominant in the more differentiated structures, the alveolar buds.     

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin''s disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.     

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.     

Clonal variations in keratin : Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.     

Molecular polymorphism of the intermediate filament protein transitin

Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.     

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.     

Phenotypic differences in subclones and long-term cultures of clonally derived rat bone cell lines

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.     

Differential expression of fibronectin in a rat histiocytoma: possible role of fibronectin in tumor cell adhesion

The specific role of fibronectin in tumor cells has been investigated using the transplantable histiocytic tumor line AK-5 [A. Khar (1986) J. Natl. Cancer Inst. 76, 871]. These cells, capable of growth as both ascites and solid tumors in rats, can be separated into four subpopulations by buoyant density centrifugation on Percoll. These subpopulations are shown to contain different amounts of fibronectin on the cell surface when studied by immunofluorescent staining followed by cytofluorometric analysis. Cells which contain high amounts of fibronectin can grow both as ascites and as solid tumors while those cells which contain low amounts of fibronectin cannot grow as solid tumors but can grow as ascitic tumors. Pretreatment of high-fibronectin-containing cells with anti-fibronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascitic tumors. These results have been discussed with reference to the specific role of fibronectin in tumor-forming cells.     

Chromosome instability in Spodoptera frugiperda Sf-9 cell line

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.     

Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence

BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.