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.     

Cell Organization and Responsiveness to Hormones in Vitro: Genesis of Domes in Mammary Cell Cultures

Domes are multicellular structures generated from confluentmonolayers of mammary epithelium under the influence of insulinand a corticosteroid hormone. The hemicyst structure and occurrencepatterns of domes suggest an in vitro analogy to organized aciniof mammary parenchyma. Two activities of dome cells, vegetativereplication of the mammary tumor virus and synthesis of casein,suggest a functional analogy between domes and acini. The corticosteroidhormone is considered the primary hormonal stimulant for domeformation. Evidence is presented that RNA and protein synthesisis required for the corticosteroid effect, as are intact activetransport functions of epithelial cells.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

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.     

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.     

Clonal growth and serial propagation of rat esophageal epithelial cells

Summary The clonal growth and serial propagation of rat esophageal epithelial cells in low serum-containing medium has been achieved without feeder layers or conditioned medium. To date, a total of four lines have been developed and maintained for as many as 40 passages in culture. Growth of the cells was possible only after modifying the culture medium (PFMR-4) by reducing the calcium concentration from 1 to 0.1 mM, and by adding low levels of dialyzed fetal bovine serum and seven growth factors; i.e. epidermal growth factor, hydrocortisone, ethanolamine, phosphoethanolamine, insulin, transferrin, and cholera toxin. Cell lines have been developed from both explant outgrowths and enzyme dissociated esophagi. The epithelial nature of the cells was confirmed by electron microscopy and immunological methods. Clonal growth studies revealed that optimal cell growth occurred in medium containing 2.4% dialyzed fetal bovine serum and 0.1 mM calcium. Calcium levels of 0.3 mM or higher caused the cells to stratify and undergo terminal differentiation. Coating the culture dishes with collagen, or a combination of collagen, fibronectin, and bovine serum albumin, increased both the cell growth rate and the colony forming efficiency. The successful long term culture of rat esophageal epithelial cells permits their use as models in studies concerned with esophageal differentiation and carcinogenesis. This investigation was supported by U.S. Public Health Service Grant CA 28950, awarded by the National Cancer Institute, Bethesda, MD.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Gangliosides stimulate dome formation in cultured canine kidney epithelial cell line (MDCK)

The effect of exogenous gangliosides on the occurrence of domes in MDCK cell cultures was investigated in view of the involvement of both dome formation and gangliosides in cell growth, differentiation and transepithelial transport. Dome formation was increased by gangliosides in medium free of fetal calf serum. Among the gangliosides tested, GM3 and GD3 isolated from porcine kidney were most active, increasing the dome number 12-17-fold. Since gangliosides from kidney were more active than those from brain and erythrocytes, the hydrophobic moiety as well as sialic acid might be involved in this activity. These results indicate that tissue-specific molecules of gangliosides function as inducers or mediators of dome formation. The mechanism probably involves adenylate-cyclase or another transmembrane biosignal-transducing system.     

Whole mammary gland organ culture: Selection of appropriate gland

Summary This report presents the results of studies on differences in the responsiveness of the different mammary glands of virgin mice in whole mammary gland organ culture. The entire second and third thoracic and the fourth inguinal mammary fat pads containing the parenchyma were excised and incubated in Waymouth's medium (MB752/1) supplemented with the hormones estradiol, progesterone, aldosterone, insulin, growth hormone, and prolactin. The rate of DNA synthesis was determined by acid-insoluble [³H]-thymidine radioactivity. Morphological measures of the extent of lobulo-alveolar development in the parenchyma were used as criteria of induction of mammogenesis in organ culture. It was evident that, after 3 and 5 days incubation in vitro, the mammary parenchyma in the second thoracic fat pad is the most responsive to the hormone-supplemented medium. This research was supported by United States Public Health Service Grant CA-11058 and Contract E-72-3212 from the National Cancer Institute.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

Growth factor responses of human arterial endothelial cells in vitro

Summary Human arterial endothelial cells were cultured in vitro for up to 40 cumulative population doublings. Culture conditions similar to those required for long-term propagation of human umbilical vein endothelial cells were employed. These included fibronectin-coated culture vessels, 5 to 20% fetal bovine serum, endothelial cell growth factor, and heparin. Thoracic aorta endothelial cells were larger than iliac artery endothelial cells. Both cell types stained positively for Factor VIII antigen by immunofluorescence. A decrease in confluent density as a function of population doubling level was correlated with the appearance of large, senescent cells in the cultures. Serum growth factors to which the arterial endothelial cells responded included insulin, transferrin, epidermal growth factor, thrombin, and somatomedins. The effect of thrombin did not require the availabilty of the active site of the protease. The effect of the somatomedins was only seen in the presence of heparin. Neither platelet-derived growth factor nor hydrocortisone induced arteiral endothelial cell proliferation. These growth factor responses were also observed on the part of human umbilical vein endothelial cells. This work was supported in part by Public Health Service grants HL01030, HL01734, and AG00599.     

Organ culture of adult rat colonic mucosa on fibrin foam

Summary A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37°C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum,l-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmosphere for culture was 95% O₂ and 5% CO₂. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [³H]thymidine and [³H]leucine into DNA and protein, [¹⁴C]glucosamine and [³H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O₂ retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland. This work was supported by the National Cancer Institute Contract N01-CP-75953 and in part by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, under Contract N01-CO-65341 with the International Union Against Cancer.     

Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts

Summary The effects of hydrocortisone and ascorbic acid on growth parameters were measured in human diploid skin fibroblasts from fetal and adult donors. In the presence of culture medium containing 10% fetal bovine serum, 0.3 μM hydrocortisone produced a 20% increase in the population growth rate and a 50 to 70% increase in the confluent density of fibroblasts from adult donors. Daily addition of 28 μM ascorbic acid also stimulated the population growth rate and cell density at confluency. The effects of hydrocortisone and ascorbic acid on the final cell density were additive. The action of hydrocortisone was restricted to cells in log-phase growth, whereas ascorbic acid affected cells in both the log and the postconfluent phases of the growth cycle. In fibroblasts from fetal donors, ascorbic acid was stimulative but hydrocortisone was not. The data suggest that whereas both compounds stimulate cell growth in an additive manner, they do so by different cellular mechanisms. This investigation was supported in part by USPHS Grants AM 02456, AM 05020 and AM 15312, and by the Kroc Foundation, No. UW 63-2986. Dr. Rowe is a fellow of the Helen Hay Whitney Foundation. Dr. Fujimoto is a recipient of a Research Career Development Award, AM 47142, from NIAMDD.     

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.     

Effect of procaine on development of ‘ridges’ and ‘domes’ in primary cell cultures from mammary glands of untreated virgin mice: The role of cell density in the occurrence of ‘domes’

Primary cell cultures from mammary glands of virgin mice that were not pretreated with hormones were subjected to: (1) procaine; (2) insulin+ prolactin +hydrocortisone; (3) a combination of (1) and (2). Procaine caused a ‘ridge’ effect similar to that of the hormones. The combination of procaine with the hormones caused a still stronger ‘ridge’ effect as well as the formation of ‘domes’. The formation of ‘domes’ is suggested to be dependent on cell density.     

Hydrocortisone stimulation of human mammary epithelial cells

Summary Rapid proliferation of mammary epithelial cells derived from biopsy specimens of human fibroadenomas was observed when medium was supplemented with ten percent fetal bovine serum and hydrocortisone (5 μg per ml⁻¹). Hydrocortisone in combination with FBS also led to a 2.5-fold increase in cell cluster attachment and subsequent colony formation. A similar effect was not observed with human serum. In contrast to fibroblast cell systems, insulin did not significantly alter cell growth. The results show that a mitogenic response to glucocorticoids by mammary epithelium may depend on the presence of factors in sera. Supported in part by NCI Contract CB-33898.     

Possible role of genetic cell variants in the viral induction of mouse mammary tumors

Summary Out of three attempts to induce neoplasia in normal C57B1 mammary epithelial cells with the mouse mammary tumor virus (MuMTV) only one presented signs of tumorigenicity. Immunofluorescence showed that virus synthesis took place in all three sublines but tumorigenicity as detected by cell aggregation viability (CAV) and transplantation into syngeneic mice failed to occur in two of them. By comparison, cells from a BALB/c spontaneous mammary tumor that do not express MuMTV were 100% tumorigenic, whereas cells from a BALB/cfC3H tumor with a 95% virus-producing cell population had a normal CAV and were tumorigenic only in 60% of the test animals. This lack of correlation suggested that many of the virus-producing cells were not neoplastic and that neoplasia might occur under virus stimulation only if a restricted population of genetic cell variants existed. Accelerated tissue culture passages of virus-free C57B1 and BALB/c normal mammary cells resulted in their spontaneous neoplasia at Passages 23 and 50 respectively; when duplicated cells cryopreserved in early passages were revived and cultivated in the same manner, neoplasia occurred at Passages 27 and 58. The similarity of the passage numbers appears to confirm the existence of genetic cell variants among the normal cell population. This investigation was supported by U.S. Public Health Service Grant R01-CA-08515 from the National Cancer Institute.     

Spontaneous transformation of a cloned cell line of normal diploid bovine vascular endothelial cells

Summary We report here the spontaneous transformation of a normal diploid bovine fetal aortic endothelial cell line. This cell line showed a period of rapid proliferation, followed by a period of declining proliferative activity, as judged by both a decline in the number of population doublings achieved from seeding to subcultivation and a decrease in the fraction of cells incorporating [³H]thymidine. During the decline in proliferation, foci of small cells appeared amid a background of larger senescent-appearing cells. The cultures then regained proliferative activity and have been maintained in our laboratory for more than 22 months. The transformants are characterized by (a) an indefinite life span, (b) a morphology that is more spindle shaped as compared to pretransformants, and (c) heteroploidy with chromosome translocations. This work was supported by the U.S. National Institute of Health (NIH) Grant AG-00378. S. D. G. is a predoctoral trainee supported by U.S. Public Health Service Grant CA-09191-06, E. H. is supported under NIH Grant AG0-2100, and W. W. N. is the S. Emlen Stokes Professor of Genetics at the Institute for Medical Research.     

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.