Hydrocortisone and progesterone regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1–10 μg/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01–1 μg/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 μg/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells. © 1995 Wiley-Liss, Inc.     

Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells

Summary Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induced ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.     

Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells

Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complement in vivo reconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.     

Adipocyte–Epithelial Interactions Regulate thein VitroDevelopment of Normal Mammary Epithelial Cells

Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complementin vivoreconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.     

Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane

We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.     

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.     

Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor. Antagonism by cyclic AMP

Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice. The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner. The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM. Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production. 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system. These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.     

Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids

In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-L1 adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation. Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-L1 adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.     

Functional role of matrix metalloproteinases (MMPs) in mammary epithelial cell development

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell (MEC) function and is remodeled by matrix metalloproteinases (MMPs). To investigate the significance and regulation of MMP activity in normal MEC, we utilized a primary culture model in which rat MEC were grown three dimensionally within a reconstituted basement membrane (RBM) in defined serum-free medium. Zymograms of culture medium demonstrated that five major gelatinases of 97, 80, 74, 69, and 65 kDa were secreted by MEC and were distinct from gelatinases of RBM origin. Based on molecular weight, p-aminophenylmercuric acid activation, immunoblotting with MMP-specific antibodies, inhibition by EDTA, a peptide containing the prodomain sequence of MMP (TMRKPRCGNPDVAN) and two synthetic MMP inhibitors (BB-94 and CGS 27023A), these were classified as inactive and active forms of MMP-9 and MMP-2. The maximal MMP activities occurred when MEC were in a rapid proliferation and branching phase and declined after they underwent functional differentiation. Known regulators of MEC growth and differentiation were evaluated for their ability to modulate gelatinase activity in primary culture. Secretion of one or both MMPs was inhibited by EGF, TGFalpha, prolactin, and hydrocortisone and stimulated by progesterone. Furthermore, the functional significance of MMPs was demonstrated since three MMP inhibitors blocked branching morphogenesis elicited by the absence of hydrocortisone. Additionally, two synthetic MMP inhibitors not only inhibited epithelial cell growth but also inhibited normal alveolar development of the MEC. Finally, these drugs were found to enhance MMP secretion from MEC, although the activity of the secreted MMPs was inhibited as long as the drug was present.     

Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.     

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA₂-arachidonic acid signalling cascade.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.