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1.
Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.  相似文献   

2.
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.  相似文献   

3.
Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.  相似文献   

4.
Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.  相似文献   

5.
Summary The concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM. The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.  相似文献   

6.
Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.  相似文献   

7.
Summary Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels with form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cel lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pincocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.  相似文献   

8.
Summary Normal and neoplastic mouse mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine. Fibroblast overgrowth was prevented and epithelial cell functions and morphology were retained in cultures maintained in, D-valine medium up to 2 months. A nonenzymatic technique was devised to dissociate epithelial cell monolayers. The combined use of this dissociation buffer and D-valine nutrient medium made it possible to passage serially normal and neoplastic mammary epithelial cells. Normal cells were derived from mammary glands of animals stimulated with exogenous hormones for various periods. The period of in vivo hormonal stimulation influenced the ability of normal mammary epithelial cells to attach and proliferate in primary and serially passaged cultures. A greater proportion of cells derived from glands following 2 to 4 weeks of hormonal stimulation were recovered after replating and showed higher labeling indices during serial passage than cells from unstimulated or 5- to 7-week stimulated groups. This investigation was supported by Grant No. CA 05388 from the National Cancer Institute and by Cancer Research Funds of the University of California.  相似文献   

9.
Summary Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c1. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32°, 34° or 37° C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32° and 34° C than at 37° C. Decreased levels of RDDP were attributed to enzyme thermolability at 37° C incubation. Research sponsored by the National Cancer Institute under Contract No. N01-CO-25423 with Litton Bionetics, Inc., and Contract No. N01-CP-33253 with the University of California.  相似文献   

10.
Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.  相似文献   

11.
Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([3H]glucosamine, [3H]fucose, [3H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.  相似文献   

12.
Summary Human mammary tissue from a female at the end of the second trimester of pregnancy was studied in organ culture in a chemically defined medium. Sampling was carried out at 1, 2 and 3 weeks. Without hormones, there was nearly total lobuloalveolar degeneration inall specimens at all times. Addition of insulin, hydrocortisone and ovine prolactin, in combination at a concentration of 5 μg per ml each, did not affect the extent of degeneration. Raising the concentration of prolactin to 50 μg per ml resulted in greatly improved lobulo-alveolar maintenance inall specimens and continued epithelial cell DNA synthesis for up to 3 weeks in vitro. This work was supported by grant no. CA11536 from the National Cancer Institute.  相似文献   

13.
Summary Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.  相似文献   

14.
Summary Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.  相似文献   

15.
AIMS: The objective of this study was to increase the acetate production by Clostridium thermolacticum growing on lactose, available as a renewable resource in the milk and whey permeate from the cheese industry. METHODS AND RESULTS: Experiments for increased acetate productivity by thermophilic anaerobes grown on lactose were carried out in batch cultures. Lactose at concentration of 30 mmol l(-1) (10 g l(-1)) was completely degraded by Cl. thermolacticum and growth rate was maximal. High concentrations of by-products, ethanol, lactate, hydrogen and carbon dioxide were generated. By using an efficient hydrogenotroph, Methanothermobacter thermoautotrophicus, in a defined thermophilic anaerobic consortium (58 degrees C) with Cl. thermolacticum and the acetogenic Moorella thermoautotrophica, the hydrogen partial pressure was dramatically lowered. As a consequence, by-products concentrations were significantly reduced and acetate production was increased. CONCLUSION: Through efficient in situ hydrogen scavenging in the consortium, the metabolic pattern was modified in favour of acetate production, at the expense of reduced by-products like ethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this thermophilic anaerobic consortium opens new opportunities for the efficient valorization of lactose, the main waste from the cheese industry, and production of calcium-magnesium acetate, an environmentally friendly road de-icer.  相似文献   

16.
Growth, expression of functional differentiation (as characterized by synthesis and secretion of milk proteins), and primary metabolism were studied for a mouse mammary epithelial cell line, COMMA-1D, in extended-batch and hollow-fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, allowing basal and apical exposure to medium. Protein production was induced in both batch and hollow-fiber cultures in hormonesupplemented medium. In batch cultures, high levels of protein production and secretion were maintained for 18 days. Once differentiation was induced, the rate of deinduction was low, even in medium containing epidermal growth factor (EGF) and serum; cells continued to express and secrete proteins for at least 12 days after prolactin and hydrocortisone were removed. Cells in both batch and hollow-fiber cultures were highly glycolytic and exhibited low rates of glutaminolysis. In batch culture on membrane inserts, cells showed polarized metabolism between the apical and basal side, maintaining significant gradients of glucose and lactate. Medium hormonal composition and subsequent differentiation affected both glucose uptake and lactate yield for COMMA-1D in batch culture. (c) 1992 John Wiley & Sons, Inc.  相似文献   

17.
Summary Effects of three adrenal corticoids on in vitro mammary differentiation were compared in neonatally estrogenized (E) and uninjected control (N) BALB/c Crgl female mice. E mice were injected with 10 μg of 17β-estradiol on each of the first 5 days of life. At 4 weeks of age, all mice were pretreated with 1 μg 17β-estradiol and 1 mg progesterone for 9 consecutive days. Groups of 10 or more entire mammary glands then were organ-cultured for 5 days at 37°C on chemically defined medium in 95% O2/5% CO2 with various combinations of hormones: mammotropin (M), somatotropin (S), insulin (I), thyroxine (T); and corticosterone (B), aldosterone (A), or cortisol (F). Differentiation followed a quantitative pattern of MSIT<BMSIT<AMSIT<FMSIT for both E and N tissues. E tissues formed more alveoli and more lobules in vitro than N tissues with each of the corticoids tested. These findings may have pathologic significance. This work was supported in part by Public Health Service Grant CA-18285 from the National Cancer Institute and by Public Health Service General Research Support Grant NIH-5-S01-RRO 5344-11.  相似文献   

18.
Summary Mice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.  相似文献   

19.
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.  相似文献   

20.
Summary Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.  相似文献   

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