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S Nandi

Publications and source records attributed to S Nandi.

At least 163 records · Page 9Linked to original sources
PubMed1987

Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture.

A two-step culture system has been developed to analyze the role of hormones in casein accumulation by mammary epithelial cells obtained from adrenalectomized and ovariectomized adult virgin mice. In the first step cells are grown inside collagen gel in medium containing insulin, epidermal growth factor (EGF), and linoleic acid for 9 days; these conditions stimulate very little casein accumulation. Following this growth phase the gels are released to float in medium containing insulin, prolactin, and linoleic acid. During this second phase the mammary cells will accumulate large amounts of casein, but only in the simultaneous presence of insulin, prolactin, and linoleic acid; in the absence of linoleic acid casein accumulation is greatly reduced. The casein accumulation is not dependent on the presence of the glucocorticoid cortisol and will occur in spite of the presence of the antiglucocorticoid agent RU 38 486. To determine if the response to cortisol observed in organ culture by other investigators might be mediated by stromal cells, epithelial cells were grown in collagen gel under fatty acid-free conditions and then cocultured with explants of mammary fat pads from adult virgin mice with or without mammary parenchyma. The cocultures were performed in fatty acid-free medium containing insulin and prolactin with or without cortisol. In the majority of experiments the mammary epithelial cells in the collagen gel accumulate more casein in the presence of cortisol than in its absence, irrespective of the presence of mammary parenchyma in the explant. Thus, mammary epithelial cells are directly dependent on insulin and prolactin for casein accumulation and indirectly dependent on cortisol by means of its effect on the stromal cells. This cortisol effect may be to cause release into the medium of linoleic acid or a metabolic product of linoleic acid from the stromal cells.

Animals↗
PubMed1987

Cultured mouse mammary epithelial cells: normal phenotype after implantation.

Mammary epithelial cells from normal virgin BALB/c mice were cultivated in vitro by the feeder cell technique developed and reported previously. These cells were cultured up to the 10th passage, equivalent to 60 cell divisions in culture. They were then tested for normality by several criteria, namely, the ability to regrow into normal mammary glands after implantation into cleared mammary fat pads of both syngeneic and nude mice, chromosome numbers, and response to mammogenic hormones. The cultured cells did form normal mammary ducts after implantation. The fraction of fat pads with ductal outgrowths as well as the size of the outgrowths was proportional to the number of cells implanted. When 10(6) cells were implanted into BALB/c mice, 83% of the fat pads contained outgrowths, filling, on the average, approximately 87% of the fat pad. More ductal outgrowths occurred from implanted cells taken from lower tissue culture passages than from high ones, and the number of outgrowths was greater in BALB/c mice than in nude mice. A small fraction of the cells in culture reacted with antibodies to casein, but there was no evidence of alpha-lactalbumin in the cells. However, ductal outgrowths from implanted cells responded to hormone stimulation of an estrogen deoxycorticosteroid pellet by forming well-developed lobulo-alveolar structures characteristic of pregnancy. Of the cells that were studied in passages 3 and 7, 85% were diploid. An abnormally growing culture in passage 10 was composed of cells in the tetraploid range. These tetraploid cells formed normal mammary ducts when implanted into animals.

Animals↗
PubMed1986

Different mitogenic and phenotypic responses of human breast epithelial cells grown in two versus three dimensions.

Human breast epithelial cells, derived from fibroadenomas, were cultured under conditions promoting growth in two-dimensions (2D) as monolayers using the collagen-coated dishes and in three-dimensions (3D) inside the collagen gel matrix. Both epidermal growth factor (EGF) and cortisol (F) were required for maximal stimulation in 3D growth, but only cortisol was required for 2D growth. The growth stimulation of exogenously added type IV collagen was no greater than that of type I as a substrate in both the 2D and 3D growth. Immunocytochemical staining, using a polyclonal actin antibody, showed homogeneous staining in all cells in 2D monolayers, whereas more restricted distribution was observed in 3D outgrowths in the collagen gel matrix. The same cells, when cultured in 2D vs 3D, elicit different responses and the original phenotypes may be better maintained in 3D.

Adenofibroma↗
PubMed1986

Effect of hormones and epidermal growth factor on the growth of the hormone-responsive 13762NF rat mammary tumor in collagen gel culture.

The hormone-responsive mammary tumor 13762NF of the F344 rat was cultured in a collagen gel matrix with the use of a serum-free medium supplemented with hormones and epidermal growth factor (EGF). Hydrocortisone (F) had the greatest effect on cell growth. EGF had no growth-promoting activity when used alone, but it had a significant effect when used with F. There was also a population of cells responsive to progesterone (P) and prolactin (PRL). P synergized with EGF as well as with PRL to promote growth. 17 beta-Estradiol alone or in combination with other hormones had no growth-promoting activity. Receptor levels in the tumor were high for glucocorticoids, intermediate for P and EGF, and low for estrogens. Metastasis in the lung and lymph node showed the same basic hormonal responses as the parent tumor. Cultured cells produced tumors with the same histopathology as the parent tumor when transplanted back into female rats; they had the same receptor levels and when placed back in culture showed a growth response to the same set of hormones. The tumor cells formed colonies that were spherical, which was different from the branching structures formed by normal mammary cells in collagen gel.

Animals↗
PubMed1986

Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel cell culture.

We investigated the effect of somatomedin C (SM-C) on the growth of mouse mammary ductal epithelial cells in collagen gel culture. Epithelial cells, isolated by collagenase digestion of whole glands, were placed into primary serum-free collagen gel cell culture for 10-12 days, during which SM-C was added alone or in combination with other growth-promoting factors. Previous work has shown that these cells require a superphysiological concentration of insulin (10 micrograms/ml) for optimum growth in serum-free medium (a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's medium) containing epidermal growth factor (EGF). When SM-C (1-250 ng/ml) alone was added to serum-free basal medium containing EGF, it stimulated growth (at concentrations greater than 25 ng/ml) to at least the same extent as insulin at 10 micrograms/ml. There was no additive stimulation of growth when optimal concentrations of insulin and SM-C were added together. The nonadditive stimulation at optimal concentrations of these hormones may indicate that the previous requirement for a superphysiological concentration of insulin for maximum growth was due to low affinity binding of insulin to the SM-C receptor. Rat insulin-like growth factor II (Collaborative Research) at 50-200 ng/ml did not stimulate growth in the presence or absence of insulin. SM-C could not stimulate growth alone. The presence of EGF or mammogenic hormones (progesterone and PRL) was required.

Animals↗
PubMed1986

Heterogeneity in the hormonal responsiveness of clones derived from the 13762NF rat mammary tumor.

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.

Adenocarcinoma↗
PubMed1985

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.

Animals↗
PubMed1985

Regulation of estrogen and progesterone receptor levels in mouse mammary epithelial cells grown in serum-free collagen gel cultures.

The effect of collagenase dissociation of virgin mouse mammary glands on the level of mammary epithelial cytosolic estrogen receptors (ER) and progesterone receptors (PR) was assessed. After cell dissociation, ER was present in mammary epithelial cells at concentrations similar to those found in the whole gland. However, PR appeared to be affected by the collagenase treatment. The regulation of ER and PR in mouse mammary epithelial cells isolated by collagenase dissociation and grown within collagen gels was then determined. After 7 days in culture under serum-free conditions inside a collagen gel, PR and, to a lesser extent ER, as characterized by high affinity binding and specificity, were present in the epithelial cells. Although at a low level, the ER were determined to be functional, since estradiol (E2) was able to promote nuclear accumulation of ER and to induce PR. PRL was able to increase cytosolic ER and PR concentrations. The combination of progesterone (P) and PRL was more effective than PRL or P alone in increasing PR. The induction of PR by P and PRL was inhibited when epidermal growth factor was present in the culture medium. Previous studies have shown that P, PRL, and epidermal growth factor, but not E2 (either alone or in combination with these factors) are able to stimulate cell proliferation in vitro. We conclude that the effects of E2 on protein synthesis and proliferation are dissociated in vitro. The difference between the effect of E2 and PRL or P on growth may be related either to the initial concentrations of their respective receptors or estrogen may stimulate growth indirectly.

Animals↗
PubMed1985

Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones.

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.

Adrenal Cortex Hormones↗
PubMed1985

Epidermal growth factor receptor levels in mouse mammary glands in various physiological states.

Experiments were undertaken to demonstrate and characterize specific receptors for epidermal growth factor (EGF) in mammary glands of female BALB/c mice in various physiological states. The results of an in vitro desaturation technique are also presented which allow estimation of the total EGF-binding sites per mg membrane protein. Binding of the ligand [125I]iodo-EGF is both time and temperature dependent. Maximum binding to the membrane is achieved after 6 h of incubation with [125I]iodo-EGF at 23 C. Scatchard analysis of equilibrium binding using membrane preparations of mammary glands from virgin mice yields two classes of high affinity receptors with Kd values of 0.8 +/- 0.1 and 5.0 +/- 0.4 X 10(-10) M and receptor concentrations of 10 +/- 1.2 and 23.5 +/- 2 fmol/mg protein, respectively. Membrane preparations of mammary tissues from cycling, gestating, and lactating mice were used to correlate cellular receptor levels to the physiological state of the animal. Beginning at weaning, there is a constant decrease in high affinity receptor level with increasing age, as well as through the early stages of both gestation and lactation. On day 10 of gestation, receptor levels increase, reaching 15.2 +/- 1.6 fmol/mg protein, followed by a decrease to 3.8 +/- 0.9 fmol/mg protein on day 10 of lactation. We conclude that membrane preparations from the mouse mammary gland contain specific high affinity receptors for EGF, and that receptor levels are characteristic of the physiological state.

Aging↗
PubMed1984

Collagen gel culture system and analysis of estrogen effects on mammary carcinogenesis.

The results obtained to date from studies dealing with the role of hormones, including estrogen, on growth of mammary epithelial cells inside the collagen gel are described. The collagen gel matrix culture system appears to be a suitable system to obtain in vivo-like effects of hormones on mammary cell in vitro. The results thus far indicate that prolactin along with progesterone or cortisol can stimulate mammary cell proliferation. Thus far, estrogen has not been found to be mitogenic in our in vitro system.

Animals↗