Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Characterization of Morphological and Cytoskeletal Changes in MCF10A Breast Epithelial Cells Plated on Laminin-5: Comparison with Breast Cancer Cell Line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, >75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

The ionic basis of oscillatory responses of skate electroreceptors

When physiological conditions are simulated, skate electroreceptors produce small maintained oscillatory currents. Larger damped oscillations of similar time-course are observed in voltage clamp. Subtraction of leakage in voltage clamp data shows that the oscillations involve no net outward current across the lumenal surface of the epithelium. The oscillations are much faster than the late outward current generated by the lumenal membranes of the receptor cells. Treatment of the basal surface of the epithelium with tetraethyl ammonium (TEA), high K, Co, or EGTA reversibly blocks the oscillations in voltage clamp, but has little or no effect on the epithelial action potential in current clamp or on the current-voltage relation. The TEA sensitivity of the oscillations indicates that they involve a potassium conductance in the basal membranes of the receptor cells. Treatment of the basal membranes with TEA and high calcium, with strontium, or with barium causes these membranes to produce large regenerative responses. Direct stimulation of the basal membranes then elicits a lumen-positive action potential whereas stimulation of the lumenal membranes elicits a diphasic action potential. Excitability of the basal membranes is abolished by extracellular Co, Mn, or La. Modulation of the lumenal membrane calcium conductance by the basal membrane conductances probably gives rise to the oscillatory receptor currents evoked by small voltage stimuli. The slower calcium-activated late conductance in the lumenal membranes may be involved in sensory accommodation.     

Multiple cellular responses to serotonin contribute to epithelial homeostasis

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers).     

The ultrastructure of the duct system in the rat extraorbital lacrimal gland

Summary The duct system of the rat exorbital lacrimal gland consists of intercalated ducts, interlobular ducts and excretory ducts. The morphological changes from one type of duct to the next are gradual. At the light microscopical level this consists of a change from a bilaminar epithelium in the intercalated ducts to an epithelium, consisting of approximately three layers — which may be pseudostratified — in the excretory ducts. The basal layer of the intercalated ducts consists of myoepithelial cells, whereas the inner epithelial cells may have both a secretory and an electrolyte transporting function. The interlobular duct epithelium contains many cells with deep infoldings of the basolateral plasma membranes and associated mitochondria, suggesting a similar function to the striated duct epithelium in salivary glands. Numerous basal cells in this epithelium have tentatively been interpreted as unusual myoepithelial cells. Nerve terminals have been observed in the ductal epithelium.This work was supported by the National Health and Medical Research Council of Australia. — We wish to thank Mrs. Eva Vasak for her expert technical assistance.     

The Expression of Psoriasin (S100A7) and CD24 Is Linked and Related to the Differentiation of Mammary Epithelial Cells

Psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is highly expressed in high-grade ductal carcinoma in situ (DCIS) and in the benign hyperproliferative skin disorder psoriasis. The gene that encodes psoriasin and many other S100 genes are located within a gene cluster on chromosome region 1q21, known as the epidermal differentiation complex. This cluster contains genes for several differentiation markers that play important roles in the terminal differentiation of the epidermis. The purpose of the present study was to evaluate the role of psoriasin in the differentiation process of mammary epithelial cells. Normal mammary epithelial cells (MCF10A) cultured in confluence and suspension, conditions known to induce psoriasin expression, demonstrated a shift towards a more differentiated phenotype indicated by an increase in the expression of the luminal differentiation markers CD24 and MUC1 and the reduced expression of the breast stem cell marker CD44. The expression of psoriasin and MUC1 was most pronounced in the CD24⁺-enriched fraction of confluent MCF10A cells. The shift towards a more differentiated phenotype was abolished upon the downregulation of psoriasin using short hairpin RNA (shRNA) and small interfering RNA (siRNA). Using specific inhibitors, we showed that psoriasin and CD24 expression was regulated by reactive oxygen species (ROS) and the nuclear factor (NF)-κB signaling pathways. While immunohistochemical analyses of DCIS showed heterogeneity, the expression of psoriasin and CD24 showed a similar staining pattern. Our findings suggest that the expression of psoriasin is linked to the luminal differentiation marker CD24 in mammary epithelial cells. Psoriasin demonstrated an essential role in the shift towards a more differentiated CD24⁺ phenotype, supporting the hypothesis that psoriasin plays a role in the differentiation of luminal mammary epithelial cells.     

Mammary gland development: Role of basal myoepithelial cells

Mammary epithelium is organized as a bilayer with a layer of luminal secretory cells and a layer of basal myoepithelial cells. To dissect the specific functions of these two major compartments of the mammary epithelium in mammary morphogenesis we have used genetically modified mice carrying transgenes or conditional alleles whose expression or ablation were cell-type specific. Basal cells are located in close proximity to mammary stroma and directly interact with the extracellular matrix (basement membrane) during all their lifespan. On the contrary, luminal secretory cells during early stages of the postnatal mammary development have only limited contacts with basement membrane and become exposed to the extracellular matrix only during late developmental stages at the end of pregnancy and in lactation. Consistently perturbation of beta1-integrin function specifically in the luminal layer of the mammary epithelium, did not interfere with mammary morphogenesis until the second part of pregnancy but led to impaired secretory differentiation and lactation. On the contrary, ablation of beta1-integrin gene in the basal mammary epithelial cells resulted in a more precocious phenotype: disorganized branching in young virgin animals and a complete arrest of lobuloalveolar development. Further, a constitutive activation of beta-catenin signaling due to expression of N-terminally truncated (stabilized) beta-catenin specifically in basal myoepithelial cells resulted in accelerated differentiation of luminal secretory cells in pregnancy, precocious postlactational involution, increased angiogenesis and development of mammary tumors. Altogether these data suggest that basal mammary epithelial cells can affect growth and differentiation of luminal secretory cells, have an impact on the epithelium-stroma relationships and, thereby, play an important role in the process of mammary morphogenesis and differentiation.     

STRUCTURE OF THE TOAD'S URINARY BLADDER AS RELATED TO ITS PHYSIOLOGY

The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.     

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.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

EGF controls the in vivo developmental potential of a mammary epithelial cell line possessing progenitor properties

The bilayered mammary epithelium comprises a luminal layer of secretory cells and a basal layer of myoepithelial cells. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells; however, their molecular characteristics and differentiation pathways are largely unknown. BC44 mammary epithelial cells in culture, display phenotypic characteristics of basal epithelium, i.e., express basal cytokeratins 5 and 14 and P-cadherin, but no smooth muscle markers. In vivo, after injection into the cleared mammary fat pad, these cells gave rise to bilayered, hollow, alveolus-like structures comprising basal cells expressing cytokeratin 5 and luminal cells positive for cytokeratin 8 and secreting beta-casein in a polarized manner into the lumen. The persistent stimulation of EGF receptor signaling pathway in BC44 cells in culture resulted in the loss of the in vivo morphogenetic potential and led to the induction of active MMP2, thereby triggering cell scattering and motility on laminin 5. These data (a) suggest that BC44 cells are capable of asymmetric division for self-renewal and the generation of a differentiated progeny restricted to the luminal lineage; (b) clarify the function of EGF in the control of the BC44 cell phenotypic plasticity; and (c) suggest a role for this phenomenon in the mammary gland development.     

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.     

v-myc alters the response of a cloned mouse mammary epithelial cell line to lactogenic hormones

Several oncogenes have now been implicated in mammary carcinogenesis. We investigated the phenotypic effects of expressing three representative oncogenes in mammary epithelial cells. v-myc (coding for a nuclear protein), v-Ha-ras (a G-protein homologue) and v-fgr (a tyrosine kinase) genes were introduced into the nontumorigenic clone 14 of the mouse mammary epithelial cell line COMMA-1D. Their effects upon growth and differentiation were determined. Anchorage-independent growth was induced by all three oncogenes with low efficiency. v-Ha-ras and v-fgr induced tumorigenicity in nude mice. The effect of oncogenes upon parameters unique to mammary epithelial cells in vitro was assayed. Both v-myc and v-fgr abolished the ability of clone 14 to grow as three-dimensional branching structures in hydrated collagen gel. v-fgr completely and v-myc partially inhibited the expression of the epithelium specific cytokeratins. Clone 14 can be induced to produce the beta-casein milk protein by the combination of the lactogenic hormones, dexamethasone, insulin, and PRL. Introduction of v-myc into clone 14 cells resulted in an estimated 50-fold increased induction of beta-casein protein and at least a 60-fold increase in beta-casein mRNA. The number of cells stained with anti-beta casein antibodies also showed a 10-fold increase after v-myc introduction. This still required the synergistic action of all three lactogenic hormones. Thus v-myc can alter the normal response of mammary epithelial cells to lactogenic hormones.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Cytokeratin profile of immunomagnetically separated epithelial subsets of the human mammary gland

Summary We have used enzymatic and immunomagnetic techniques for the physical separation of the basal and luminal epithelium of the human mammary gland. Immediately after tissue dissociation and cell sorting, we have examined the steady-state CK composition of these cells individually by direct two-dimensional gel electrophoresis of intermediate filament extracts. Our results demonstrate that cytokeratins typical of simple and stratified epithelial cells are simultaneously expressed by both mammary epithelial subclasses in vivo. Moreover, the entire spectrum of cytokeratins seen in vivo is also maintained in short-term cultures of human mammary epithelium. A comparison of the data obtained by direct cytokeratin analysis with published indirect immunolocalization studies is presented. The ability to isolate purified epithelial subsets from normal human breast tissue by a simple immunomagnetic procedure demonstrated here can facilitate the development of relevant model systems for studying the regulatory components in growth, differentiation and malignant transformation of the human breast.