Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI-PMEC

To express recombinant proteins in the spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) currently established in our laboratory, a chemically synthesized DNA fragment encoding the anticoagulant hirudin was used to construct a mammalian expression vector under the control of the goat beta-casein regulatory sequence. The vector, named pGB562/Hi, was transfected into the SI-PMEC cells to yield pGB562/Hi/SI-PMEC. The pGB562/Hi/SI-PMEC cells expressed recombinant hirudin only when they were differentiated into functional structures by growth on a Matrigel-coated petri dish supplemented with the lactogenic hormone prolactin. The differentiated pGB562/Hi/SI-PMEC cells produced about 0.5-0.6microg of recombinant hirudin/mg of total cellular protein. These results suggest that the established SI-PMEC cells have pharmaceutical potential to inducibly express bioactive heterogeneous proteins.     

Establishment of Mammary Gland Model In Vitro: Culture and Evaluation of a Yak Mammary Epithelial Cell Line

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.     

Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions

This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.     

Hugl1 and Hugl2 in Mammary Epithelial Cells: Polarity,Proliferation, and Differentiation

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.     

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties

Summary EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H₁B clone form branching cords devoid of a central lumen, K₃A₃ cells from cords enclosing small multifocal lumina, and J₃B₁ cells form large cavitary structures containing a wide lumen. I₃G₂ cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Be1a cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell-lined cavitary structures, except H₁B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesis.     

Evidence that aberrant expression of tissue transglutaminase promotes stem cell characteristics in mammary epithelial cells

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells.     

Establishment and characterization of a caprine mammary epithelial cell line (CMEC)

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.     

Effects of the extracellular matrix on fetal choroid plexus epithelial cells: changes in morphology and multicellular organization do not affect gene expression.

We have developed a primary culture system for fetal mouse choroid plexus epithelial cells which maintains their differentiated phenotype. When grown on a reconstituted basement membrane substrate (Matrigel) epithelial cells formed aggregates which became embedded in the matrix and developed into characteristic and highly reproducible multicellular vesicular structures. These vesicles consisted of a squamous layer of epithelial cells with extensive attachment to the matrix substrate, surrounding a fluid-filled lumen. Electron microscopy showed that cells comprising these vesicles had a high degree of membrane specialization and polarized morphology which in many respects mimicked the in vivo morphology. Biochemical analyses demonstrated that under these culture conditions the tissue-specific pattern of gene expression of fetal choroid plexus epithelium was maintained. After 6 days in culture these cells contained approximately the same amount of transthyretin mRNA as the 12.5-day choroid plexus in vivo, and the level of total RNA per cell, which is proportional to the protein synthetic capability of the cells, was also maintained. The pattern of protein secretion was also very similar to that generated by fetal mouse choroid plexus cells in vivo. In contrast choroid plexus epithelial cells attached poorly to collagen I gels. Heterogeneous aggregates were formed in which cell-cell interactions were more extensive than cell-substrate interactions, and in no cases was a central lumen observed. Cells on the surface of large aggregates showed some evidence of membrane polarization, while the majority of cells in the cultures exhibited little evidence of polarized morphology. Despite the striking difference in morphology and multicellular organization these cells still expressed high levels of transthyretin mRNA and maintained the same pattern of protein synthesis as cells cultured on Matrigel. These results indicate that the basement membrane is important for the organization of choroid plexus epithelial cells into a functional epithelium in vitro and thus presumably the maintenance of the integrity of the blood-brain barrier in vivo. In contrast to several other epithelial systems which have been studied, the type of extracellular matrix does not appear to directly influence tissue-specific gene expression by choroid plexus epithelial cells. Thus the level of gene expression is not dependent on the cytoarchitecture and multicellular organization of this cell type.     

Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma-Dbeta cell line

A mouse mammary epithelial cell line with morphogenetic properties in vivo, Comma-Dbeta, was used to isolate and to characterize mammary progenitor cells. We found that a homogeneous cell population expressing high surface levels of stem cell antigen 1 (Sca-1) was able to give rise in vivo to ductal and alveolar structures comprising luminal secretory and basal myoepithelial cells. Unlike the Sca-1(high), the Sca-1(neg/low) cell population displayed a reduced morphogenetic potential. The Sca-1(high) cells presented moderate CD24, high CD44 and alpha6 integrin surface levels, expressed basal cell markers p63, keratins 5 and 14, but no luminal and myoepithelial lineage markers. In culture, the Sca-1(high) cells generated identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self-renewal. Plated at clonogenic density in Matrigel, Sca-1(high) cells formed spheroids that included luminal and myoepithelial cells. Thus, the isolated Sca-1(high) basal cells possess several features of stem/progenitor cells, including specific markers, self-renewal capacity, and the ability to generate the two major mammary lineages, luminal and myoepithelial. These data provide evidence for the existence of basal-type mouse mammary progenitors able to participate in the morphogenetic processes characteristic of mammary gland development.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.     

OMEC II: A new ovine mammary epithelial cell line

We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow rapidly on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO-1, a tight-junction associated protein, but negative immunostaining for an anti-vimentin antibody. In confluent cell monolayers ‘domes’ became visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end-buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of β-lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation.

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic "cobblestone" morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in beta-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) alpha s- and beta-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

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.     

Insulin-like growth factor binding protein (IGFBP)-5 is upregulated during both differentiation and apoptosis in primary cultures of mouse mammary epithelial cells

We have previously demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) is upregulated following treatment of the mouse mammary epithelial cell line HC11 with lactogenic hormones (dexamethasone, insulin, and prolactin-DIP). In addition, we have also shown that IGFBP-5 is upregulated in mammary epithelial cells in vivo during involution of the rodent mammary gland. We have, therefore, postulated that there may be a dual regulation of IGFBP-5 expression during the temporally separated processes of differentiation and apoptosis of mammary epithelial cells. To test this hypothesis further, we have used a phenotypically differentiated model, which comprises primary cultures of mouse mammary epithelial cells grown on a layer of EHS (Engelbreth-Holm-Swarm) extracellular matrix. We show that lactogenic hormone treatment (hydrocortisone, insulin, and prolactin-HIP) of these cultures induces the upregulation of IGFBP-5 thus replicating the results obtained with the HC11 cell line. In addition, following the induction of apoptosis in primary cultures of mammary epithelial cells by treatment with TGFbeta-3, IGFBP-5 expression is also upregulated. In parallel with this upregulation of IGFBP-5, there is also an increase in the levels of cleaved caspase-3, a well-characterized marker of cellular apoptosis. These findings confirm previous in vivo work demonstrating an increase in IGFBP-5 expression during involution of the mouse mammary gland. When HC11 cells are cultured under serum-free conditions (a well-characterized apoptotic insult in cell culture), there is also an increase in cleaved caspase-3 levels. Unexpectedly, in the presence of TGFbeta-3, caspase-3 levels are attenuated. In the presence of DIP, caspase-3 levels are also decreased in HC11 cells. As described previously, TGFbeta-3 inhibits beta-casein synthesis in HC11 cells. In the HC11 cell line (in contrast to primary cultures of mammary epithelial cells), there is no evidence for TGFbeta-3 induction of IGFBP-5 under either serum-free or DIP-supplemented conditions. We believe our data with primary cultures of mammary epithelial cells support the hypothesis of dual regulation of IGFBP-5 expression during both differentiation and apoptosis in the mammary gland and emphasizes the importance of using appropriate cell culture models to investigate such phenomena in this tissue. We discuss the possible implications of our observations in relation to the physiological processes of pregnancy, lactation, and involution in the mammary gland and the associated changes in mammary epithelial cell function.     

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland : Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

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.