Effects and interactions of epidermal growth factor, insulin, hydrocortisone, and estradiol on the cloning of human tumor cells

We investigated the effects and interactions of epidermal growth factor (EGF), insulin, hydrocortisone, and estradiol on the growth of 18 freshly obtained human tumors in our human tumor stem cell assay (HTSCA) cultured at a reduced serum concentration (8.5% ml). All possible combinations of these four supplement factors were added to the assay to determine the ability of each component to enhance colony formation. We found that hydrocortisone was the most effective single supplement in stimulating colony growth in the HTSCA. Supplementation with insulin, estradiol, or both had some growth-promoting effect but not as great as hydrocortisone. Moreover, the addition of insulin, estradiol, or both often demonstrated a negative interaction with hydrocortisone. EGF supplementation alone; in dual combination with insulin, estradiol, or hydrocortisone; or in combination with estradiol and insulin in the assay did not significantly increase colony formation. However, EGF added to the cultures containing hydrocortisone with insulin and/or estradiol significantly increased colony formation and reversed the negative effect of insulin and estradiol on hydrocortisone activity. Thus, under conditions of our assay, the most effective combination in promoting colony growth contained all four factors.     

Enhancement of α-lactalbumin-like activity in mammary explants from pregnant rats in the absence of exogenous prolactin

Mammary explants from pregnant rats showed a progressive increase in α-lactalbumin activity during culture with insulin, hydrocortisone and prolactin. Unexpectedly, culture with only insulin and hydrocortisone produced a similar rate of increase of α-lactalbumin-like activity, but this increase commenced about 24 hr later. The delay suggests that the enhanced activity effected by insulin and hydrocortisone is not a reflection of carry-over of endogenous mammotrophic hormones. Insulin plus hydrocortisone did not stimulate casein or fatty acid synthesis by pregnancy tissue, and did not enhance α-lactalbumin-like activity in virgin rat mammary explants. Enhancement of this activity by insulin plus hydrocortisone in pregnant tissue was constant over a wide range of glucocorticoid concentrations, but was inhibited by progesterone. Available evidence indicates that the active factor in extracts from insulin-hydrocortisone-explants is a heat-stable protein which is either α-lactalbumin itself, or another molecule with similar specifier properties.     

Effect of hydrocortisone on ACTH, growth hormone, insulin and glucose in the blood of bilaterally adrenalectomized patients

This study is aimed at elucidating the mechanism of paradoxical rise in plasma ACTH levels in response to glucocorticoids, observed by several authors in bilaterally adrenalectomized patients with Cushing's disease. Six control subjects and fourteen patients bilaterally adrenalectomized for Cushing's disease were given a dose of 200 mg hydrocortisone sodium succinate by 3-5 mm i.v. injection. Plasma ACTH (in 6 patients), serum cortisol, growth hormone (GH) and insulin and blood glucose levels were estimated at 0, 30, 60, 90, and 120 minutes. The administration of hydrocortisone significantly suppressed plasma ACTH levels only at 60 min. In one case a slight rise in ACTH level during the test was observed. A significant fall in blood glucose levels was found only in the adrenalectomized patients. No significant changes in serum insulin and GH levels were noted. The possible mechanisms are discussed, especially the potential role of transient glucose deficiency in the pathophysiology of plasma ACTH increase in response to hydrocortisone in the bilaterally adrenalectomized patients.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

Progesterone and prolactin are both required for suppression of the induction of rat alpha-lactalbumin activity

Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.     

Comparison of collagen gels and mammary extracellular matrix as substrata for study of terminal differentiation in rabbit mammary epithelial cells

Mammary epithelial cells were prepared by collagenase digestion of tissue from mid-pregnant rabbits and cultured for up to 6 days on either collagen gels or an extracellular matrix prepared from the same tissue. The behaviour of the cells in serum-supplemented medium containing combinations of insulin, prolactin, hydrocortisone, estradiol and progesterone were monitored by measuring rates of casein synthesis, lactose synthesis, DNA synthesis and protein degradation. After 6 days, epithelial cells on floating collagen gels showed substantial increases in casein synthesis and DNA synthesis over freshly-prepared cells, following a decline during the first 3 days when the collagen gels are contracting. The optimum hormone combination for casein synthesis was insulin + prolactin + hydrocortisone, whereas for optimum DNA synthesis the additional presence of estradiol and progesterone was required. Cells on extracellular matrix showed increased rates of both casein synthesis and DNA synthesis by day 6 in the presence of insulin + prolactin + hydrocortisone, with additional estradiol + progesterone having an inhibitory effect. Whereas on day 2 rates of intracellular protein degradation were generally lower in cells on extracellular matrix, by day 6 rates of protein degradation were lowest in cells cultured on collagen gels with insulin + prolactin + hydrocortisone. In all cases, rates of lactose synthesis fell to low levels as the culture proceeded. Pulse-chase labelling of freshly-prepared cells with [32P]orthophosphate in medium containing serum and insulin + prolactin + hydrocortisone demonstrated that newly-synthesized casein was degraded during its passage through the epithelial cell. The influences of the collagen gels and extracellular matrix and of the hormone combinations on epithelial cell differentiation and secretory activity are discussed.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

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.     

Multihormonal regulation of thyroglobulin production by the OVNIS 6H thyroid cell line

The hormonal regulation of thyroglobulin production has been studied using a clone of the ovine thyroid cell line: OVNIS 6H. 3 among the 6 hormones proposed for serum replacement are required for an optimal thyroglobulin production; insulin, hydrocortisone and thyrotropin. Insulin alone stimulates thyroglobulin production. The presence of insulin is also required to observe hydrocortisone and TSH stimulations. Newborn calf serum inhibits thyroglobulin production. The best conditions for optimal thyroglobulin expression and TSH responsiveness are obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.     

Effect of hydrocortisone and insulin on (14C)-glycine and (3H)-methionine incorporation into spleen proteins in alloxan-diabetic rats

The intensity of labelled amino acid incorporation into spleen total proteins of albino rat with experimental diabetes was studied as influenced by hydrocortisone, insulin and both hormones simultaneously. Hydrocortisone inhibits and insulin stimulates the [14C] glycine and [3H] methionine incorporation into total proteins of alloxan-diabetic rat spleen. Under simultaneous administration of both hormones the inhibitory hydrocortisone effect is allayed by insulin. It is suggested that an increased level of glucocorticoid hormones in blood is one of the reasons of protein metabolism disturbance under diabetes mellitus.     

Murine islet cryopreservation and corticosteroids: functional studies

Knowledge of protective effects of corticosteroids on traumatized cells prompted us to test the potential benefit of islet cryopreservation in the presence of hydrocortisone. Neonatal murine islets were isolated by collagenase, followed by 2- to 3-day tissue culture. Precryopreservation glucose-stimulated (50-500 mg/dl) insulin release was 25-388% above basal (mean = 113%) in 18/20 fresh islet preparations. Subsequent freezing was done in RPMI 1640 medium plus 10% (v/v) heat-inactivated fetal calf serum and 10% (v/v) Me2SO with or without 1 mg/ml hydrocortisone at 0.25 degrees C per minute in a programmed freezing system, to -80 degrees C, and stored for greater than 60 days at -196 degrees C. Thawing, by transfer to room air, was followed by dilution, 4x (v/v), in 4 degrees C RPMI plus 10% protein, after which glucose-stimulated insulin release was reassessed, showing 56-280% response over basal in 3/8 steroid-treated preparation and 20-220% response in 3/10 control preparations. Basal insulin release was 0.72 ng/microgram protein/hr in fresh islets (N = 20) and 0.22 ng/microgram protein/hr after freeze-thawing. We conclude that functional islet survival by this method is approximately 30% and that hydrocortisone did not improve viability.     

A unique and essential role for insulin in the phenotypic expression of rat mammary epithelial cells unrelated to its function in cell maintenance

Mammary explants from pregnant rats can be induced in regard to casein synthesis and alpha-lactalbumin activity when cultured in the presence of hydrocortisone, prolactin and levels of insulin approaching physiological concentrations. No detectable induction occurs in the absence of insulin. Although epidermal growth factor and multiplication stimulating activity, in the presence of hydrocortisone, can maintain the initial level of NADH-cytochrome c reductase as well as insulin, neither can substitute effectively for insulin in the induction of the milk proteins. Proinsulin, nerve growth factor, platelet-derived growth factor and fibroblast growth factor are also ineffective substitutes for insulin in this regard. Whereas prolonged tissue exposure to multiplication stimulating activity, hydrocortisone and prolactin does not result in induction of alpha-lactalbumin activity, subsequent addition of insulin leads to prompt response. The results suggest that the ability of insulin to function as a unique, essential factor in the induction of rat milk proteins is independent of its cell-maintenance activity. Thus, in addition to its well established functions in metabolic processes, insulin appears to play a vital role in certain developmental processes.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Influence of loach egg- and embryo-extract on the activity of lactate dehydrogenase in the presence of insulin

Studies of the pig muscle lactate dehydrogenase (LDH) activity were permormed after incubating the enzyme solution with extracts of loach (Misgurnus fossilis) eggs and embryos, which were subjected to the influence of insulin hydrocortisone as well as to insulin combination with actinomycin D, cycloheximide or puromycin. The insulin alone is established to decrease the inactivating ability of the investigated extracts on the lactate dehydrogenase activity, when antibiotics removed to a considerable extent the influence of hormone. In the eggs and embryos there are proteins activating and stabilizing the LDH molecule. The level of LDH activation under the influence of the eggs and embryos extracts subjected to the insulin action was decreased. The addition of hydrocortisone to the medium for the incubation of eggs and embryos does not affect significantly the LDH-inactivating ability of their extracts.     

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

The regulation of transformed phenotypes was studied in newly isolated preadipose cell lines which were established after infection with simian virus 40 tsA58 dl2009. The clonal cell lines isolated exhibited most of the characteristics typical of transformed cells. The transformants, however, were able to differentiate into adipocytes in the presence of low calf serum (0.5%) and a combination of several hormones, including hydrocortisone and insulin. Treatment with insulin alone stimulated the growth of these cells but did not induce lipid accumulation without added hydrocortisone. The effect of hydrocortisone was accompanied by a restoration of growth control in the transformants after they reached high cell density. The blot hybridization analysis of cellular DNAs digested by restriction enzymes revealed that simian virus 40 genomes were integrated at multiple separate sites at which a head-to-tail oligomeric insertion took place. Large T antigen was synthesized in growing cells but was regulated at high cell density when cells were committed to differentiate by glucocorticoids. These results suggest that the glucocorticoid hydrocortisone is capable of restoring growth regulation at high cell densities to simian virus 40-transformed preadipose cell lines.