Selective enhancement of the induction of alpha-lactalbumin activity in rat mammary explants by epidermal growth factor

Epidermal growth factor (EGF) enhances the induction of alpha-lactalbumin in mammary explants from pregnant and virgin rats in the presence of insulin (I), hydrocortisone (F) and prolactin (P). EGF also enhances the prolactin-independent induction of alpha-lactalbumin in tissue from pregnant rats and evokes prolactin-independent induction of alpha-lactalbumin in mammary tissue from virgin rats in the presence of I and F. Casein synthesis and galactosyltransferase activity are unaffected by EGF in the IFP-system, and are not induced in the IF-EGF-system. Multiplication stimulating activity, nerve growth factor, fibroblast growth factor and platelet-derived growth factor do not mimic the selective effects of EGF on rat alpha-lactalbumin. These influences of EGF on the differentiation of isolated rat mammary tissue are compared with those on mouse and rabbit tissue studied previously.     

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.     

Prolactin-induced alpha-lactalbumin activity in mammary explants from pregnant rabbits. A role for epidermal growth factor and glucocorticoids.

Exogenous prolactin alone can induce alpha-lactalbumin activity in rabbit mammary explants. Under these conditions, exogenous corticosol has no effect. However, low levels of epidermal growth factor (EGF) can markedly inhibit the induction by prolactin, and this inhibitory effect, in turn, can be prevented by cortisol. The steroid can, in fact, convert EGF from a potent inhibitor into an agent which enhances the induction. None of the other growth factors tested inhibits induction of alpha-lactalbumin activity by prolactin, and no other type of steroid tested overcomes the effect of EGF. The results suggest that, under physiological conditions, glucocorticoid may be essential for induction of alpha-lactalbumin activity in the rabbit in order to override the effect of serum EGF.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

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.     

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.     

Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline

Depletion of proline from insulin, hydrocortisone, and prolactin-containing medium prior to incubating virgin mouse mammary explants prevents both DNA synthesis and functional differentiation in the mammary epithelial cells; however, DNA synthesis in the mammary stroma and total incorporation of radioactive amino acids into total protein appears to continue without hindrance. Removal of glycine instead of proline had no deleterious effect on either DNA replication in the hormone-stimulated epithelium or in its functional differentiation. Functional differentiation was determined by the induction of casein and alpha-lactalbumin synthesis in the insulin, hydrocortisone, and prolactin (IFPrl)-treated explant cultures. As a control, the induction of mouse mammary tumor virus (MMTV) gene expression, a corticosteroid-regulated function, was also measured. Neither the absence of proline or glycine prevented the glucocorticoid stimulation of MMTV gene expression. In contrast to mammary tissue from virgin mice, explants from nonpregnant primiparous mice responded fully to IFPrl stimulation with respect to DNA, casein, and alpha-lactalbumin synthesis in medium depleted of proline. These data suggest that the uncommitted epithelium of virgin mouse mammary glands requires the presence of exogenous proline in order to respond to lactogenic hormonal signals. We have demonstrated earlier that DNA synthesis is a prerequisite of functional differentiation in virgin mouse mammary explants (Smith and Vonderhaar, 1981, Dev. Biol., 88:167-179; Vonderhaar and Smith, 1982, J. Cell Sci, 53:97-114), although cytological differentiation proceeded unencumbered in explants prevented from synthesizing DNA. Here, without proline, neither cytological nor functional differentiation can be induced; this suggests that proline provides an essential metabolic interlock in the acquisition of lactogenic hormone responsiveness in uncommitted mouse mammary tissue.     

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.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

Effects of hormonal growth factors on colony formation and chemosensitivity of human tumors in agarose

Summary Because low plating efficiencies of most human cancers severely limit the number of successful chemosensitivity tests that can be performed, we studied the growth-enhancing effects of hormonal growth factors on a variety of solid tumors. Dose-response studies with progesterone and estradiol indicated no benefit from adding these substances to the culture medium. This was true whether progesterone or estradiol was used alone or in the presence of other hormones. By contrast, epidermal growth factor (EGF) at concentrations from 10 to 100 ng/ml increased colony numbers up to 10-fold. Although insulin, hydrocortisone, and EGF used alone could either stimulate or inhibit the growth of specific tumors, the combination of all three (hormone mixture or HM) was always at least as good and usually better than any individual component in increasing cloning efficiency. HM-supplemented medium gave significantly increased colony counts in 41/46 tumors. Sensitivity to anticancer drugs was not changed in 63 paired drug tests.     

Effect of hormones and EGF on proliferation of rat mammary epithelium enriched for alveoli : An in vitro study

Virgin rat mammary epithelium enriched for alveoli were embedded in a collagen gel matrix to study the direct effect of mammogenic hormones and epidermal growth factor (EGF) on their growth over a 12-day culture period. Serum-supplemented medium alone caused a 3- to 4-fold increase in cell number, whereas medium containing insulin, prolactin, progesterone, cholera toxin and serum caused a 15-fold increase. Cultures resulting from this substantial cell number increase consisted of large, smooth-bordered epithelial colonies with relatively few (< 1%) single cells surrounding them. An equal increase in cell number was obtained when progesterone was replaced by hydrocortisone in the above-mentioned medium, but these cultures contained predominantly single spindle-shaped cells with a few small epithelial colonies. The smooth-bordered epithelial colonies consisted solely of mammary epithelial cells, since they contained thioesterase II, an enzyme found exclusively in mammary epithelium. The identity of the single spindle-shaped cells remains to be determined. The addition of EGF to serum or serum, hormone and cholera toxin-supplemented medium did not enhance the proliferative effect of these factors on the alveolar-enriched population.     

Prolactin induction of casein mRNA in organ culture. A model system for studying peptide hormone regulation of gene expression

The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice.

Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Pituitary-induced lactation in mammary gland explants from the pregnant tammar (Macropus eugenii): a negative role for cyclic AMP

1. alpha-Lactalbumin and casein have been isolated from tammar milk. 2. alpha-Lactalbumin was induced in mammary explants by culture with anterior pituitary. 3. Casein was induced maximally in the presence of a physiological concentration of prolactin alone. 4. Progesterone did not inhibit the prolactin-induced synthesis of casein, alpha-lactalbumin, galactosyltransferase or fatty acids. 5. Both dibutyryl cAMP and a combination of cholera toxin and IBMX did significantly inhibit the induction of casein and alpha-lactalbumin. 6. Progesterone withdrawal is not a component of the lactogenic trigger in this marsupial but cAMP may be a common intracellular signal for negative control of lactogenesis in both marsupials and eutherians.     

Regulation of c-fos expression in primary culture of guinea pig glandular epithelial cells stimulated by growth factors and estradiol

The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation

We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females. PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ(+)) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ(+) cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. LacZ(+) cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ(+) mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse epithelial cell fates.