B--> micro+ micro- in the two-Higgs-doublet model.
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Journal Article↗
Biomedical subjects
S Nandi
Publications and source records attributed to S Nandi.
Basic fibroblast growth factor stimulates the growth and inhibits casein accumulation in mouse mammary epithelial cells in vitro.
The effects of basic fibroblast growth factor (bFGF) on the growth and differentiation of mouse mammary epithelial cells in serum-free collagen gel culture were examined. Epithelial cells obtained from virgin or midpregnant mice grew when bFGF was added to medium containing either insulin at a concentration greater than or equal to 1 microgram/ml or somatomedin-C (Sm-C) at 150 ng/ml. This growth-promoting effect is of the same magnitude as, and additive with, the growth-promoting effect of epidermal growth factor (EGF) or mammogenic hormones. The sensitivity of the cells to EGF or mammogenic hormones was not altered by exposure to bFGF. The progeny cells resulting from growth stimulation by bFGF are capable of accumulating casein upon subsequent stimulation by prolactin (PRL), but accumulate less casein than cells grown in response to EGF. bFGF also appears to reduce casein accumulation if it is added to the cultures at the same time as PRL.
Animals↗
Turnover of epidermal growth factor binding sites in mouse mammary epithelial cells.
An in vitro method of studying epidermal growth factor (EGF) receptors in mouse mammary epithelial cells in serum-free collagen gel culture has been developed. Binding of EGF averaged 108 +/- 19 fmol/mg DNA in cells isolated from freshly dissociated virgin mammary tissue. Initial binding values were maintained in cells cultured in the presence of 0.1 ng/ml EGF, but decreased in either 0 ng/ml or 10 ng/ml EGF. Addition of either chloroquine (100 microM) or ammonium chloride (10 mM) to the culture medium increased receptor levels 10-fold. Cycloheximide (0.1 microgram/ml), ouabain (3 mM), and actinomycin D (5 x 10(-2) micrograms/ml) each decreased receptor levels, in some cases by as much as 80%. Both methylamine (10 mM) and dinitrophenol (0.1 mM) had no significant effect. These studies suggest that the net level of EGF receptors in these target cells is the result of an equilibrium between synthesis and degradation. The difference between the effects of the compounds tested on either receptor degradation or synthesis in comparison to cell growth, may be indicative that receptor degradation is not linked to cell proliferation.
Animals↗
Phospholipids containing polyunsaturated fatty acyl groups are mitogenic for normal mouse mammary epithelial cells in serum-free primary cell culture.
Epithelial cells obtained by collagenase digestion of mammary glands from virgin BALB/c mice were cultured in collagen gels in serum-free basal medium containing insulin (10 micrograms/ml), to which lipids or growth factors were added. Synthetic phospholipids were added as liposomes. Dilinoleoyl phosphatidic acid or phosphatidylserine or epidermal growth factor stimulated multifold growth. The optimum mitogenic effect of the phospholipids was dependent upon the presence of a polyunsaturated fatty acid esterified to the sn-2 position of the glycerol moiety. Dilinoleoyl phosphatidylcholine also stimulated growth but was generally less stimulatory than phosphatidylserine or phosphatidic acid, and phosphatidylethanolamine did not stimulate growth. Studies using phospholipids radiolabeled in either the sn-2 fatty acyl group or the glycerol backbone showed that the relative effect of phospholipids on growth did not correlate directly with the extent of their incorporation into cellular lipid, indicating that phospholipid turnover was the more important determinant for mitogenesis. Analysis of phosphatidic acid-stimulated growth suggested that both cAMP-dependent and cAMP-independent pathways were involved. Thus, mitogenic phospholipids stimulate proliferation by activating (directly or indirectly) multiple growth-regulatory pathways in mammary epithelial cells.
1-Methyl-3-isobutylxanthine↗
Spermidine is necessary for, but not the only mediator of, linoleic acid-stimulated alpha-casein accumulation in mouse mammary epithelial cells.
Mammary epithelial cells obtained from virgin mice were induced to accumulate alpha-casein in serum-free two-stage collagen gel culture with insulin, PRL, and linoleic acid. Omission of either PRL or linoleic acid drastically reduces alpha-casein accumulation. Spermidine addition to insulin-containing medium in either the absence or presence of linoleic acid does not stimulate alpha-casein accumulation. We conclude from this that spermidine can not be the sole mediator of PRL action. Spermidine also will not replace linoleic acid for alpha-casein accumulation if the fatty acid is omitted from the culture medium and will not increase casein accumulation when supplemented into PRL- and linoleic acid-containing medium; thus, it is not the sole mediator of linoleic acid action. However, the spermidine synthesis inhibitor methylglyoxal-(bis)-guanylhydrazone does inhibit alpha-casein accumulation in a concentration-dependent and spermidine-recoverable manner in cells stimulated by PRL and linoleic acid. We could not detect changes in polyamine levels in response to any of the medium supplements used in this investigation. Spermidine is, thus, at least a required comediator of the alpha-casein synthesis induction, although its role remains enigmatic.
Animals↗
Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels.
Mammary epithelial cells from 3-4-month-old BALB/c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbecco's Modified Eagle's Medium/Ham's F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [3H]dimethylbenz[a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [3H]DMBA to water and organosoluble metabolites. By 72 h, 77-94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-,3-, or 4-hydroxydimethylbenz[a]anthracene, the hydroxymethyl derivatives, 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[a]anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[a]anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.
9,10-Dimethyl-1,2-benzanthracene↗
Proliferative effects of insulin and epidermal growth factor on mouse mammary epithelial cells in primary culture. Enhancement by hydroxyeicosatetraenoic acids and synergism with prostaglandin E2.
Linoleate metabolism via the cyclooxygenase pathway enhances the proliferation of mammary epithelial cells in serum-free culture in the presence of epidermal growth factor and insulin (Bandyopadhyay, G.K., Imagawa, W., Wallace, D., and Nandi, S. (1987) J. Biol. Chem. 262, 2750-2756). Prostaglandin E2 (PGE2) can fully substitute for linoleic acid provided endogenous hydroxyeicosatetraenoic acids (HETEs, lipoxygenase metabolites) are available. The PGE2 effect is partial if lipoxygenase activity is inhibited by nordihydroguaiaretic acid. Any combination of two HETEs out of three tested (5-, 12-, and 15-HETEs) stimulates growth synergistically with PGE2; and together (i.e. PGE2 + HETEs), they completely substitute for linoleate. In the absence of PGE2, maximal stimulation cannot be attained with HETEs. Exogenous 5-HETE, compared with 12- or 15-HETE, is preferentially incorporated by the mammary epithelial cells, and about 25-30% of it is retained esterified in phospholipids. The cellular level of nonesterified, free HETE is low. Radioimmunoassay revealed that the concentrations of 12- and 15-HETEs in the culture media (with or without added linoleate) were always higher than that of 5-HETE. Both intra- and extracellular free HETEs are rapidly metabolized by the cells. Since these cells are capable of producing eicosanoids from linoleate, periodic supplementation of the cultures with linoleate allows maintenance of higher HETE and PGE2 levels. Thus, it appears that not only are HETEs short-lived in the cell cultures, but cells handle 5-HETE differently than 12- and 15-HETEs. Whatever may be the pathways of interaction, synergism between HETEs and PGE2 seems to explain how linoleate stimulates the growth of mammary epithelial cells in the presence of epidermal growth factor and insulin.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Angular distribution of W-boson pairs at a heavy Z resonance.
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Journal Article↗
Growth stimulation by PGE2 and EGF activates cyclic AMP-dependent and -independent pathways in primary cultures of mouse mammary epithelial cells.
Mammary epithelial cells from virgin Balb/c mice were isolated by collagenase digestion and cultured within collagen gels in serum-free basal medium containing insulin (10 micrograms/ml). Previous work has shown that linoleate or its metabolite, prostaglandin E2 (PGE2), stimulate the growth of these cells only in the presence of a growth stimulant such as epidermal growth factor (EGF). Since PGE2 can stimulate cyclic AMP (cAMP) production, the role of cAMP in linoleate and EGF-stimulated growth was examined. The cAMP phosphodiesterase inhibitor, IBMX (0.1 mM), was found to augment growth when cells were cultured in the presence of both EGF and linoleate or PGE2, but not either factor alone. These results indicated that EGF does not stimulate proliferation via cyclic AMP mediated events but could synergize with cAMP events if cAMP levels were elevated by PGE2. When assayed in cells plated on top of collagen-coated culture dishes, cellular cyclic AMP levels were stimulated by PGE2, but only marginally by EGF. Although the stimulation of endogenous cAMP by PGE2 and IBMX was insufficient to stimulate growth in the absence of EGF, exogenous dibutyryl-cAMP (greater than 100 micrograms/ml) was able to do so showing that a sustained, and high level of cAMP (greater than 100 micrograms/ml) could stimulate growth in insulin-containing basal medium. EGF was capable of enhancing the cellular sensitivity to dibutyryl-cAMP but the converse was not observed. cAMP stimulation of growth was dependent upon a superphysiological concentration of insulin (10 micrograms/ml) or a physiological concentration of somatomedin-C. These results indicate that the proliferation of mouse mammary epithelial cells can be stimulated separately or in synergism by cAMP-dependent or -independent events.
1-Methyl-3-isobutylxanthine↗
Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1.
Normal rat mammary epithelial cells were cultured within a rat tail collagen gel matrix formed under improved conditions for controlling pH and osmolarity. Under these conditions, growth can be maintained for up to 3 weeks with a 10- to 15-fold increase in cell number. The cells grow in response to prolactin, progesterone, epidermal growth factor, and cholera toxin, in a medium of DME: Ham's F12 supplemented with BSA and insulin at 10 micrograms/ml. When the insulin concentration was reduced to more physiological levels (10 ng/ml) the cells did not grow. However, at these more physiological concentrations it could be shown that insulin had a concentration-dependent effect on the maintenance of the cells with an optimum concentration around 25 ng/ml. The cells could be maintained in hormone-supplemented medium with low levels of insulin in a quiescent state for up to 14 days. The high levels of insulin needed for optimal growth could be replaced by insulin-like growth factor 1 (IGF-1) at much lower concentrations (25-50 ng/ml). The superphysiological level of insulin required for optimum growth is probably due to its acting weakly through an IGF-1-mediated growth-promoting mechanism. Insulin's effect on cell maintenance occurs at physiological levels and may better reflect its role in mammary cell growth.
Animals↗
Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization.
Mammary epithelial cells from adult virgin mice have been cultured within collagen gels in totally serum-free medium containing either epidermal growth factor or the mammogenic hormones, progesterone and prolactin, or prolactin alone. The cellular organization, differentiation and cell-type composition of the colonies from the three culture conditions were assessed by transmission electron microscopy and light-microscope immunocytochemistry. The epithelial cells form branching duct-like structures and, when exposed to mammogenic hormones, assume a secretory morphology (including casein micelles) similar to that seen in the early to mid-pregnant mouse.
Animals↗
Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea.
High-efficiency neoplastic transformation of mouse mammary epithelial cells in primary collagen gel culture was induced by N-methyl-N-nitrosourea (MNU). Mammary epithelial cells, isolated from virgin BALB/c mice, were embedded within collagen gels and grown in a serum-free medium containing prolactin, progesterone, and linoleic acid. The cells were then treated with MNU on day 3 of culture and subsequently at weekly intervals for up to 4 weeks. Eleven to 14 days after the final carcinogen treatment, the cells were removed from the collagen gels and injected into the cleared mammary fat pads of syngeneic hosts to assay for transformed cell populations. A single exposure or multiple exposures of these cells to MNU was effective in inducing tumorigenic cells that produced palpable tumors as early as 6 weeks after transplantation. Two treatments with MNU (100 micrograms/ml) were optimal for neoplastic transformation and produced tumors in 79% of the injected fat pads. All the tumors originated at the site of injection and had extensive central necroses. Histological examination indicated that the tumors were mammary carcinomas. Secondary transplantation of tumor pieces into intact mammary glands produced palpable carcinomas of the same histology within 1-8 weeks. Control cells cultured for the same periods of time as MNU-treated cells produced only ductal outgrowths that were morphologically similar to those found in the mammary glands of adult virgin hosts. This system provides a distinct means to study the mechanism of mammary neoplastic transformation at cellular and molecular levels.
Animals↗
The lack of effect of phenol red or estradiol on the growth response of human, rat, and mouse mammary cells in primary culture.
Normal and neoplastic mammary cells from both human and rodent sources grow in culture in response to a number of hormones and growth factors. However, with the exception of a few human tumor lines, a consistent growth-promoting effect of estrogens on mammary cells has not been observed. Mammary cells can be shown to respond to other hormones and factors, such as PRL, hydrocortisone, and epidermal growth factor. Recent observations suggest that the pH indicator dye phenol red, found in most media, may be masking any exogenous estrogenic effects by acting as a weak estrogen. To test this possibility, we reexamined the effects of estradiol (E2) and the antiestrogen keoxifene on the growth of normal human, mouse, and rat mammary cells in the absence of phenol red. Primary cultures of these mammary cells were grown within a rat tail collagen gel matrix in a serum-free medium made up of Ham's F-12 and Dulbecco's Modified Eagles' medium (1:1) with and without phenol red. The medium was supplemented with various hormones and growth factors. These supplements were selected for each cell type to produce a variety of conditions from nongrowing to rapidly growing. The effects of E2 (10(-10)-10(-8) M, keoxifene (10(-9)-10(-6) M), and phenol red on growth under these various conditions were examined. Phenol red had no effect on growth, and its absence did not restore a response to E2. Keoxifene, in the presence or absence of E2, also had no effect on growth. Although E2 had no effect on growth, it was able to induce a 150% increase in the progesterone receptor levels in normal mouse mammary cells in culture, indicating that the cells retain their capacity to respond to E2. This work supports the idea that the effect of estrogens on growth in vivo may be mediated through some other factor(s).
Animals↗
Exclusive decays and masses of the B mesons.
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Journal Article↗
Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor.
Linoleic acid, arachidonic acid, prostaglandin E1, and prostaglandin E2 stimulated the proliferation of mammary epithelial cells in serum-free primary cultures only in the presence of epidermal growth factor. Linoleate-stimulated growth was manifest later in culture when proliferation, initiated by epidermal growth factor only, reached a plateau while linoleate-supplemented epidermal growth factor cultures continued to proliferate. The cultures in the plateau phase of growth could be restimulated to grow by adding either linoleic acid or prostaglandin E2 to the media. While the linoleate response could be abolished by the cyclooxygenase inhibitor, indomethacin, prostaglandin E2-stimulated growth remained unaffected. Linoleic acid was metabolized to arachidonic acid and prostaglandin E2, both in the growing and resting cultures. Proliferating cells metabolized linoleate and prostaglandin E2 extensively so that neither the fatty acid nor prostaglandin E2 accumulated in large quantities in the proliferating cultures. The concentrations of prostaglandin E2 in growing cultures supplemented with linoleic acid were much higher than in cultures without it. These results suggest that the metabolism of linoleic acid leading to prostaglandin production, not its contribution to membrane polyunsaturation, is necessary for sustained growth of mammary epithelial cells in the presence of epidermal growth factor.
Alprostadil↗
In vitro transformation of mouse mammary epithelial cells grown serum-free inside collagen gels.
Mammary epithelial cells from 4-month-old virgin BALB/c mice were cultured inside collagen gels in the following serum-free media: Dulbecco's modified Eagle's medium:Hams's F-12 (1:1) supplemented with: insulin (10 micrograms/ml), bovine serum albumin (5 mg/ml), and epidermal growth factor (5 ng/ml); insulin, bovine serum albumin, progesterone (0.05 microgram/ml), and prolactin (1 microgram/ml); insulin, bovine serum albumin, progesterone, prolactin, and linoleic acid (10 micrograms/ml). Cells proliferated in all these media. The cells were treated with 0.01 micrograms/ml of 7,12-dimethylbenz(a)anthracene or 100 micrograms/ml of N-nitroso-N-methylurea on day 3 of culture and, subsequently, at 1-week intervals for 3-6 weeks. Tetradecanoylphorbol acetate (0.1 micrograms/ml) was added to selected cultures. The cultures were maintained for up to 9 weeks; the cells were then removed from the collagen gels, placed in monolayer culture for 2 days, and removed from monolayer culture, and 5 X 10(5) cells were transplanted to each of the gland-free mammary fat pads of 3-week-old female mice. Approximately 10 weeks after transplantation, the transplanted mammary fat pads were examined for outgrowths. Cells that were not treated with carcinogen and cultured for up to 9 weeks in different serum-free media and transplanted to the gland-free mammary fat pad produced only ductal outgrowths similar in morphology to the ducts of the virgin host's mammary glands. Six treatments with 7,12-dimethylbenz(a)anthracene, of cells grown in the presence of epidermal growth factor, induced 31% spindle cell tumors, 17% ductal hyperplasias, and 5% lobuloalveolar hyperplasias. Cells that were grown in epidermal growth factor and treated three times with N-nitroso-N-methylurea produced 23% ductal hyperplasias and 17% lobuloalveolar hyperplasias. Cells grown in the presence of progesterone and prolactin and treated three times with 7,12-dimethylbenz(a)anthracene produced up to 23% lobuloalveolar hyperplasias and 12% ductal hyperplasias. Three treatments with N-nitroso-N-methylurea of cells grown in progesterone- and prolactin-containing media produced a maximum of 50% lobuloalveolar hyperplasias and 33% ductal hyperplasias. The lobuloalveolar hyperplasias have the characteristics of the precancerous hyperplastic alveolar nodules found in mouse mammary tumorigenesis. The in vitro carcinogen-induced lobuloalveolar hyperplasias were transplantable, maintained their lobuloalveolar morphology in virgin hosts, and produced carcinomas.
9,10-Dimethyl-1,2-benzanthracene↗
Human breast epithelial cells in serum-free collagen gel primary culture: growth, morphological, and immunocytochemical analysis.
Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum-free medium. Response to various additives has been analyzed for growth-promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct-like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re-created in the collagen gel system starting from small clumps of cells.
Adenofibroma↗
Estrogen receptor in dissociated and cultured human breast fibroadenoma epithelial cells.
Estrogen binding was measured by a whole cell receptor assay in epithelial cells isolated from 20 premenopausal patients with breast fibroadenomas. A high affinity specific binding for estrogens was detected in the epithelial cells isolated from all 20 fibroadenomas. A relationship between estrogen binding and the phase in the menstrual cycle of the patient has been observed. Cell culture experiments using serum-free medium have also shown that estrogen binding can be augmented by cortisol.
Adenofibroma↗