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.     

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.     

Temporal effect of prolactin on the activities of lactose synthetase, alpha-lactalbumin, and galactosyl transferase in mouse mammary gland explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.     

Proteomic discovery of genistein action in the rat mammary gland

Genistein, the primary isoflavone component of soy, consumed in diet during the prepubertal period suppresses chemically induced mammary cancer in rats. The current study used two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology to identify proteins responsible for genistein breast cancer protection In Vivo. Female offspring were exposed via lactating dams treated with 250 mg genistein/kg AIN-76A diet from days 1 to 21 postpartum (prepubertal period). Mammary glands were collected at 21 and 50 day of age and subjected to 2-DE/MS and immuno-blot analyses. Twenty-three proteins were determined to be differentially regulated (p < 0.05) and identified using 2-DE, followed by MALDI-TOF/TOF or LC-ESI-MS/MS. Five of these proteins were validated by immuno-blots. Annexin A2 was significantly increased at 21 days yet found to be decreased at 50 days. Fetuin B was found to be unchanged at day 21 but increased at day 50. Phosphoglycerate kinase 1 (PGK1) was unchanged at day 21 but decreased at day 50. Gelsolin was increased at day 21 but not at day 50. Protein disulfide-isomerase A3 (PDIA3) was decreased at day 21 and unchanged at day 50. Also, we found that vascular endothelial growth factor receptor 2 (VEGF-R2) and epidermal growth factor receptor (EGF-R) were decreased in mammary glands of 50-day-old rats treated prepubertally with genistein. This study demonstrates the usefulness of proteomics for the discovery of key proteins involved in signaling pathways to understand genistein mechanisms of action in breast cancer prevention.     

Effect of curcumin on the production of nitric oxide by cultured rat mammary gland.

We have hypothesized that one aspect of the antitumor activity of curcumin (diferuloylmethane) during the promotion stage of mammary gland tumorigenesis may be linked to reduction of free radicals (Inano et al., Carcinogenesis, 20: 1011-1018, 1999). Nitric oxide (NO) has been found to inflict damage on important biomolecules, and the overproduction of NO in diseases may be implicated in carcinogenesis and tumor progression. We have reported that the presence of three isoforms of nitric oxide synthases (NOS) and NO generation in the mammary gland correlate with the mammary gland development and mammary carcinogenesis. We, therefore, investigated the inhibitory activity of curcumin for the production of NO in rat mammary glands by using an organ culture system to validate the effectiveness and usefulness of curcumin in the pathophysiology of the mammary gland. A diced mammary gland (approximately 3 mm cubes) from the inguinal part of a female Wistar-MS rat treated with estradiol and progesterone was cultured with 2 ml of 5% FCS/DMEM in the presence or absence of LPS (0.5 microg/ml) for 2-3 days. Curcumin ( approximately 100 microM) was added at the same time to the LPS-treated cultures. In some experiments, curcumin was added to the culture after the LPS had been washed out. The NO production was significantly increased (by almost 20-fold compared to the control) by the addition of LPS to the culture system. This enhancement of NO production by LPS was reduced to 76 and to 56% by addition of 30 and 100 microM curcumin, respectively, to the culture. When LPS was eliminated from the culture after prestimulation for 1 day, the production of NO by the mammary gland dropped off, although some NO was still detectable. Curcumin did not further inhibit the production of NO by the prestimulated mammary gland after the elimination of LPS from the culture. The inducible nitric oxide synthase (iNOS, 122 kDa) and endothelial nitric oxide synthase (eNOS, 152 kDa) isoforms were detected in the mammary gland extracts at the end of the organ culture. The quantity of iNOS was apparently increased in the gland treated with LPS, while the eNOS expression was clearly diminished. Curcumin (100 microM) obviously suppressed the iNOS expression in the mammary glands cultured with LPS, and a recovery in the eNOS expression was observed. On the other hand, curcumin exhibited scavenging activity for the NO released from N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC 12), a NO donor compound, in the coincubation mixture. These results indicate that curcumin has the ability to inhibit iNOS induction by LPS in the mammary gland and to scavenge NO radicals, which might explain, at least partly, its therapeutic properties in inflammation of the mammary gland.     

Enzymes of mercapturic acid production in rat mammary gland.

1. The enzymes glutathione S-transferase, gamma-glutamyl peptidyltransferase and dipeptidase, which participate in the detoxification pathway through mercapturic acid production, were measured in rat mammary gland during pregnancy and lactation. 2. Mammary-gland concentration of reduced glutathione showed, concomitantly with the enzyme activities, a significant increase during lactation. 3. The mammary-gland glutathione S-transferase exhibits characteristics quite similar to those described for the liver and kidney enzymes with respect to substrates, isoenzymes, molecular weight and probenecid and bilirubin inhibition. 4. In view of these similarities, mammary-gland glutathione S-transferase may play the same role as a cytoplasmic organic-anion receptor proposed for the hepatic enzyme. It may also represent a detoxification pathway for protecting the mammary tissue during the lactogenic cycle.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.     

Regulatory factors on parathyroid hormone-related peptide production by primary culture of lactating rat mammary gland.

Parathyroid hormone-related protein (PTHrP) is a major cause of humoral hypercalcemia of malignancy, but has also been widely found in fetal and adult non-neoplastic tissues. Lactating mammary gland has been shown to produce large amounts of PTHrP, and high levels of PTHrP have been measured in milk. We have examined the influences of several substances on the secretion of two different forms of PTHrP by primary cultures of mammary cells isolated from lactating rats to examine the regulatory mechanisms of PTHrP production by mammary cells. Primary cultures of mammary cells seeded at a density of 10(5) cells per 35 mm culture dish were grown on collagen gels. First, after cells were left 24 hours for attachment and incubated in 2 % FCS containing medium with for 12 hours, PTHrP (1 - 87) secretions were measured in conditioned medium with hormone supplementation for 1, 24 and 48 hours. Progesterone (10(-7) - 10(-5) mol/l) significantly suppressed PTHrP (1 - 87) secretion in a dose-dependent manner (p < 0.01), while 17beta-estradiol had no influence on PTHrP (1 - 87) secretion. Prolactin, a known stimulator of PTHrP expression in vivo, had no effect in this in vitro model. Second, PTHrP (1 - 34) secretion levels from confluent lactating mammary cells for 24 hours were evaluated. The same results were obtained in the case of PTHrP (1 - 87) secretion from non-confluent cells. Furthermore, dexamethasone (10(-6) mol/l) significantly suppressed PTHrP (1 - 34) secretion (p < 0.01). These results suggest that PTHrP production from the lactating mammary gland is suppressed by progesterone as well as dexamethasone. Progesterone dramatically falls after delivery, thus possibly accelerating PTHrP production by lactating mammary glands and resulting in considerable amounts of PTHrP secreted into the milk.     

Effects of cyclic nucleotides on lipid biosynthesis in mouse mammary gland explants

The effects of dibutyryl cAMP, 1-methyl-3-isobutyl xanthine (MIX), cGMP, dibutyryl cGMP, and 8-bromo cGMP on the rate of lipid synthesis in mouse mammary gland explants were studied. Dibutyryl cAMP at 10(-4) M selectively inhibited the effect of prolactin on the rate of [14C]acetate incorporation into lipids. At 10(-3) M, dB-cAMP inhibited the effects of insulin, insulin plus cortisol, and prolactin. The phosphodiesterase inhibitor, MIX, inhibited both basal and prolactin-stimulated incorporation rates in a concentration-dependent fashion. These data suggest an inhibitory role for cAMP in the regulation of lipogenesis in the mammary gland. Cyclic GMP, db-cGMP, and 8-bromo cGMP were all without effect on either basal or prolactin-stimulated incorporation rates. Therefore, it appears that cGMP, by itself, is not involved in the regulation of lipogenesis in the mammary gland.     

Effects of arachidonic acid on RNA metabolism in mammary gland explants of mice

Arachidonic acid stimulated ³H-uridine incorporation into RNA in mammary gland explants of mice in a manner similar to that of prolactin. The onset of the effects of both prolactin and arachidonic acid occurred following a 2–4 hour lag period. Further, effects of maximally stimulatory concentrations of these agents were nonadditive. Finally, indomethacin abolished the effects of both prolactin and arachidonic acid. A tenable hypothesis to explain these data is that the action of prolactin in the mammary gland may be mediated by an increased availability of arachidonic acid followed by a subsequent enhanced rate of formation of the prostaglandins.     

Effects of arachidonic acid on RNA metabolism in mammary gland explants of mice

Arachidonic acid stimulated ³H-uridine incorporation into RNA in mammary gland explants of mice in a manner similar to that of prolactin. The onset of the effects of both prolactin and arachidonic acid occurred following a 2–4 hour lag period. Further, effects of maximally stimulatory concentrations of these agents were nonadditive. Finally, indomethacin abolished the effects of both prolactin and arachidonic acid. A tenable hypothesis to explain these data is that the action of prolactin in the mammary gland may be mediated by an increased availability of arachidonic acid followed by a subsequent enhanced rate of formation of the prostaglandins.     

Binding and production of insulin-like growth factor-I in rat mammary gland.

1. Mammary tissue from pregnant rat presents low and high affinity IGF-I functional receptors. 2. Mammary explants from pregnant and lactating rats secrete IGF-I and its production was related to the developmental stage of the gland. 3. An inverse relationship between IGF-I production and tissue binding capacity was observed.     

Effect of prolactin on phosphate transport and incorporation in mouse mammary gland explants

Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of (32)PO(4) was six times higher than in the culture medium after a 4-h treatment with (32)PO(4). Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated (32)PO(4) uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited (32)PO(4) uptake. All three lactogenic hormones together effected the greatest stimulation of (32)PO(4) uptake. Similar hormone effects were observed with regard to (32)PO(4) incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of (32)PO(4) uptake and incorporation occurred 8-12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO(4) uptake was determined to be approximately 7.7 mM x h(-1) x l cell water(-1); the apparent Michaelis-Menten constant was approximately 3-5 mM. The PRL effect on (32)PO(4) uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of (32)PO(4) uptake and incorporation into macromolecules in cultured mouse mammary tissues.     

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.     

Effect of iodide-deficiency on rat mammary gland.

When rats are kept iodide-deficient, atrophy and necrosis takes place in the mammary gland and areas of dysplasia and atypia are seen. Administration of estradiol to iodide-deficient rats stimulates cell division in the gland and leads to the formation of alveoli. Continued stimulation by estradiol produces changes in the newly-formed alveolar cells. Their nucleoli are altered and show a separation of components. Ribosomes and lipid droplets increase and the cells synthesize large vacuoles containing protein. The secretion of great quantities of this material into areas of the tissue where regressive changes have occurred undoubtedly contributes to the formation of cysts within the gland. The present findings indicate that iodide-deficiency alters the structure and function of mammary gland alveolar cells and makes them highly sensitive to stimulation by estradiol.