Quantitative characterization of domes in primary mouse mammary epithelial tumor cell cultures

Summary Dome populations from primary cultures of mouse mammary tumor cells were quantitatively studied in regard to size, distribution, density and the area occupied by light-diffusion photography and image analysis. The effects of fetal bovine serum, insulin and hydrocortisone were analyzed. Quantitative characterization documented dome diameter (mode diameter 0.26 to 0.52 mm), dome area occupied (average 23%, maximum 38.7%), and density (average 78 domes per cm², maximum 117 domes per cm²) for standard culture conditions. Insulin and hydrocortisone had a primary effect on dome density whereas 15% fetal bovine serum had a minimal effect. However, insulin and hydrocortisone had a synergistic optimal effect on dome population. Time-lapse cinematography revealed that the dome population is not static, but a very dynamic one. Domes underwent irregular pulsations of expansion and contraction. Dome enlargement was either by a series of expansions and contractions, by lateral involvement of other cells, or by coalescence of two or more domes. Domes have been considered to be the in vitro counterpart of the in vivo acinus of the mouse mammary gland. However, quantitative dome population characterization has not been available. Dome analysis by light-diffraction photography and image analysis lends itself towards correlative studies of domes and their differentiative products. Supported in part by Public Health Service Contract NO1-CP 61013 within the Virus Cancer Program of the National Cancer Institute and by Public Health Service Training Grant CA05245 from the National Cancer Institute.     

Differentiation and morphogenesis of mammary cells in vitro

Cells of a mammary cell line isolated from a DMBA-induced rat mammary carcinoma undergo differentiation in vitro. A reversible differentiation leads to the formation of two types of microstructure (domes and ridges); this paper is concerned with the mechanism of dome formation. This differentiation is initiated by inducers, some of which are generated in the cultures and act locally; their effect is strongly dependent on cell concentration and requires hydrocortisone. There are, in addition, exogenous inducers as well as inhibitors. In the pathway to dome formation important roles are played by cAMP (probably both intracellular and extracellular), the organization of the cytoskeleton, and the Thy-1 antigen. The pathway and the significance of the phenomenon for mammary gland development are discussed.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

Differentiation in human endometrial cells in monolayer culture: Dependence on a factor in fetal bovine serum

Human epithelial cells of the Ishikawa endometrial line can be stimulated to differentiate and form multicellular structures in 4–5 day-old monolayer cultures by the addition of a protein factor from fetal bovine serum. Multicellular structures become obvious over an 18–30-h period as the cells enlarge, separate from the dish, and form domes. These structures are similar to those that result from polarization in other epithelial cell lines. Ishikawa dome formation appears to be a multistage process. The appearance of enlarged differentiated cells is detected within hours of adding fetal bovine serum; these enlarged cells lift off the surface of the dish within 6–8 more hours. Domes are observed about 24 h after the addition of fetal bovine serum. Sometimes dome cells migrate into a “bud-like” structure that extends out from the dome. Differentiation of the domes is dependent on a factor from fetal calf serum that behaves similarly to a very large protein or complex of proteins, greater than 300 kd. Progesterone appears to enhance the formation of domes but does not elicit dome formation in the absence of serum factor.     

Why there was a useful plausible analogy between geodesic domes and spherical viruses

In 1962, Donald Caspar and Aaron Klug published their classic theory of virus structure. They developed their theory with an explicit analogy between spherical viruses and Buckminster Fuller's geodesic domes. In this paper, I use the spherical virus-geodesic dome case to develop an account of analogy and deductive analogical inference based on the notion of an isomorphism. I also consider under what conditions there is a good reason to claim an experimentally untested analogy is plausible.     

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.     

Lysosomotropic agents and inhibitors of cellular transglutaminase stimulate dome formation, a differentiated characteristic of MDCK kidney epithelial cell cultures

Dome formation is a manifestation of transepithelial fluid transport in cell culture, a differentiated characteristic of transporting epithelia. A dramatic increase in numbers of domes in confluent MDCK kidney epithelial cell cultures was noted after addition of Friend cell inducers such as hexamethylane bisacetamide (HMBA) (Lever, 1979b). In the present study, we show that primary amines such as methylamine, ethylamine, and dansyl cadaverine also stimulate dome formation. These compounds largely prevented the marked decrease in numbers of spontaneously occurring domes which occurred when cultures were switched from medium containing 10% serum to medium containing serum concentrations below 0.2%. Many of these primary amines are not only lysosomotropic agents but also potent inhibitors of transglutaminase activity when assayed in MDCK cell extracts, at concentrations correlating with those effective in stimulation of dome formation. Other lysosomotropic agents such as chloroquine and secondary and tertiary amines stimulated dome formation yet did not inhibit transglutaminase. Induction of domes by HMBA differed in several properties from that stimulated by amines and did not involve fluctuations in transglutaminase activity. These findings suggest that lysosomal functions modulate serum stimulation of dome formation in epithelial cells by a pathway distinct from that triggered by HMBA.     

Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

Retinoic acid modulates dome formation by MDCK cells in defined medium

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K-1). After 4-5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7 days with 3 x 10(-7) M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 x 10(-3) microns 2 in area, whereas in control medium the majority of the domes were between 0 and 9 x 10(-3) microns 2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K-1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1-independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K-1 lacking PGE1. This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1-independent MDCK cells. Dibutyryl cAMP-resistant MDCK cells, which normally do not form domes in medium K-1, were also studied. Remarkably, the dibutyryl cAMP-resistant MDCK cells were observed to form domes at a significant frequency when medium K-1 was supplemented with retinoic acid. However in medium K-1 lacking PGE1, an effect of retinoic acid on dome formation by dibutyryl cAMP-resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP-resistant MDCK cells to form domes in medium K-1 has previously been attributed to their decreased cAMP-dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP-dependent protein kinase or activation of a separate pathway.     

Electrical currents flow out of domes formed by cultured epithelial cells

Domes are localized areas of fluid accumulation between a cultured epithelial cell monolayer and the impermeable substratum on which the cells are cultured in vitro. Dome formation has been documented in a variety of epithelial cell lines that retain their transepithelial transport properties in vitro. However, it is not known whether domes are predominantly areas of specific active transport, or, alternatively, are predominantly areas of relative weak attachment to the culture surface. In the present study we adapted a vibrating microelectrode, which can detect small currents flowing in extracellular fluid, to determine if current was flowing into or out of domes and thereby to determine if domes were specialized areas of active transport. We used alveolar type II cells as the main epithelial cell type because they readily form domes in vitro and because they transport sodium from the apical to the basal surface. We found that electrical current flowed out of domes. The direction of the current was independent of the size of a dome, of the age of an individual dome, and of the number of days in primary culture for alveolar epithelial cells. This current was inhibited by amiloride and ouabain and was dependent on sodium in the medium. We made similar observations (outward current from domes which is blocked by amiloride and by sodium substitution) with domes formed by the Madin-Darby canine kidney cell line. The data support the hypothesis that sodium is transported across the entire monolayer and leaks back mainly through the domes. We conclude that domes in epithelial monolayers are not predominantly special sites of active transport but are more likely simply areas of weak attachment to the substratum.     

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.     

Glucose deprivation results in the induction of glucose-regulated proteins and domes in MDCK monolayers in hormonally defined serum-free medium

Madin-Darby canine kidney (MDCK) cells spontaneously form dome-like structures in vitro, a phenomenon which has been proposed to be indicative of cellular differentiation. This study indicates the existence of a correlation between the induction of domes and of glucose-regulated proteins during glucose starvation. When MDCK monolayers were glucose deprived, domes appeared very rapidly. After only 3 h of glucose deprivation domes appeared in 69% of the microscope fields. The level of expression of glucose-regulated proteins (grps) as well as domes was examined over a 6-h time interval of glucose deprivation. Both grp 76 and 97 were induced over this time interval, with grp 76 being the more readily detectable. The level of induction of grp 76 as a function of time was quantitated by means of densitometry measurements. The induction of domes was examined in parallel with the induction of grp 76. The results indicated that the induction of grp 76 and domes occurs with a similar time course. The effect of glucose deprivation on the initial rate of ouabain-sensitive Rb+ uptake was also examined. Within the first 4 h of glucose deprivation, the initial rate of ouabain-sensitive Rb+ uptake did not differ significantly in glucose-deprived and control MDCK monolayers. These observations indicate that unlike the case with other methods of dome induction (e.g., treatment with either prostaglandin E1 (PGE1) or hexamethylene bis-acetamide (HMBA] glucose deprivation does not affect the Na+K+ ATPase activity of MDCK monolayers. These observations suggest that PGE1, HMBA, and glucose deprivation affect dome formation in MDCK monolayers by means of distinct mechanisms.     

Effect of aldosterone on dome formation by MDCK mono-layer

Effect of aldosterone on the dome formation in the reconstructed MDCK cell epithelia was studied. MDCK cells derived from dog kidney are assumed to be originated from distal tubules or collecting ducts. When cultured to a confluency, these cells formed a epithelial layer with many domes which contained fluid transported from the apical to the basolateral surface through this layer. Aldosterone at a concentration of 10(-8) to 10(-6) M increased the number of domes dose-dependently, probably through a receptor mediated process, since the dome formation induced by this hormone was completely abolished in the presence of spironolactone. This study primarily disclosed that the dome formation in MDCK cells was stimulated by aldosterone, probably through a receptor mediated mechanism.     

Dome formation in the human colon carcinoma cell line Caco-2 in culture. Influence of ouabain and permeable supports

We studied formation of domes in cell monolayers of the human colon carcinoma cell line Caco-2 which has been shown to exhibit signs of enterocytic differentiation and transport properties. After a 24 hr incubation with 4 X 10(-8) M ouabain, the number of domes seen on Caco-2 cell monolayers grown on plastic dishes was not significantly altered. After a 90 min preincubation with ouabain, 86rubidium uptake by Caco-2 cells was inhibited by ouabain, indicating that the cells have an ouabain-sensitive Na+, K+-ATPase, while dome formation was unaffected by ouabain. Domes were observed in Caco-2 cell monolayers grown on Nuclepore filters when the pore size was 0.015 micron but not when it was 0.030 micron. Our results suggest that dome formation in the Caco-2 cell line could be independent of Na+, K+-ATPase activity and might be due to accumulation of molecules having an effective hydrodynamic radius comprised between 0.015 and 0.030 micron.     

Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation: Dissociation from effects on transepithelial resistance and cyclic AMP levels

Changes in transepithelial electrical resistance and cyclic nucleotide levels were monitored accompanying chemical induction of domes in a clonal subline of MDCK kidney epithelial cells. Confluent cell monolayers grown on nitrocellulose filters exhibited a relatively high mean transepithelial resistance (387 ohms · cm²). Hexamethylene bisacetamide, a potent inducer of dome formation (Lever, 1979b), stimulated significantly increased transmonolayer resistance as well as elevated levels of intracellular cyclic AMP. By contrast, dimethylformamide, an equally potent inducer of dome formation in MDCK cells, did not appreciably alter either resistance values or cyclic nucleotide levels. These results suggest that induction of dome formation in epithelial cell cultures by compounds generally known as inducers of differentiation may involve multiple and separate mechanisms.     

Differentiation of epithelial Na+ channel function. An in vitro model

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R(t)) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel gamma-subunit (gamma-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of alpha- and beta-ENaC and of alpha1- and beta1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.     

Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro

Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called "domes." MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly identical to those of noninfected normal mammary epithelium in primary culture. Cell to cell growth regulatory signals were modified in cultures of nonalveolar tumor cells wherein evidence of overgrowth is documented.     

Transepithelial transport in cell culture. A theoretical and experimental analysis of the biophysical properties of domes.

Dissociated cells of transporting epithelia, when cultured on an impermeant substratum, form polarized monolayers frequently characterized by the presence of domes. If the assumption is made that the monolayer exhibits a uniform stretch modulus of elasticity and tension of cell-dish adhesion, Ta, then biophysical properties of the epithelium can be predicted. We have shown that for such epithelia, domes should (a) have circular bases, (b) be sections of spheres with a constant height to radius, h/r, ratio, (c) have a dome-wall tension, Tw, that is constant, and (d) have a dome volume that is a function of radius alone. Additionally, a Laplace equation derived for this geometry predicted the hydrostatic pressure from within to outside domes as a decreasing function of radius alone. By microscopy, domes had predominantly circular bases and were found to be sections of spheres with a constant height, h, to radius, r, ratio of 0.684. Using the Laplace equation derived for this geometry and measurements of delta P and r, the tension of cell-dish adhesion, Ta, and dome-wall tension, Tw, were found to be constants of 6.60 and 7.08 torr, respectively. Combining the constants for Ta and h/r ratio, and the fact that domes are sections of spheres, delta P and dome volume were shown to be known functions of radius alone. In addition, the modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2.     

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues

Summary Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.Abbreviations DE dome epithelium - DEL dome epithelial lymphocytes - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - PBS phosphate-buffered saline - PBS-CaMg PBS containing calcium and magnesium ions - UEA Ulex europaeus agglutinin I The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of DefenseIn conducting the research described in this report, the investigators adhered to standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USA     

Taphonomy and habitat preference of North American pachycephalosaurids (Dinosauria,Ornithischia)

The traditional view of North American pachycephalosaurids holds that their domes are typically worn, as though they had undergone extensive fluvial transport, and that these animals therefore inhabited upland environments (e.g. piedmont/intermontane settings) relative to where their remains are typically found. Using a statistically robust sample of domes from Alberta, Canada, we subject these hypotheses to various forms of quantitative testing and show that: (1) domes typically exhibit minimal rounding; (2) dome roundness does not positively correlate with distance from the presumed intermontane origin; and (3) pachycephalosaurid remains are not relatively more abundant in intermontane than in alluvial or coastal plain palaeoenvironments. These findings contradict the traditional view of North American pachycephalosaurid dome taphonomy and support the argument that pachycephalosaurids frequented alluvial and coastal lowlands.