NHERF1/EBP50 controls lactation by establishing basal membrane polarity complexes with prolactin receptor.

The development of the lactating mammary gland is a complex multifactorial process occurring in mammals during pregnancy. We show here that this process requires NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ERM-binding phosphoprotein 50) expression and that successful lactation depends on NHERF1 allele copy number, with rates of 50 and 20% in NHERF1(+/-) and (-/-) mice, respectively. The prolactin receptor (PRLR)-STAT5 signaling provides the central axis triggering the differentiation of secretory mammary alveolar cells. In successfully lactating glands, NHERF1 is massively upregulated and forms complexes with PRLR, but also with ß-catenin, E-cadherin and ezrin at the alveolar basal membrane, establishing basal polarity. In NHERF1-deficient glands, the basal polarity is disrupted, the PRLR levels and basal membrane localization are abolished, and the downstream STAT5 activation collapses with consequent reduction of milk protein synthesis. NHERF1/EBP50, a protein deregulated in breast cancer, thus emerges as an important physiological mediator of milk secretion, by engagement of PRLR in multimeric complexes at the alveolar basal membrane with subsequent network activation leading to cell differentiation.

Mtbp haploinsufficiency in mice increases tumor metastasis.

Mdm2 inhibits the function of the p53 tumor suppressor. Mdm2 is overexpressed in many tumors with wild-type p53 suggesting an alternate mechanism of loss of p53 activity in tumors. An Mdm2-binding protein (MTBP) was identified using a yeast two-hybrid screen. In tissue culture, MTBP inhibits Mdm2 self-ubiquitination, leading to stabilization of Mdm2 and increased degradation of p53. To address the role of MTBP in the regulation of the p53 pathway in vivo, we deleted the Mtbp gene in mice. Homozygous disruption of Mtbp resulted in early embryonic lethality, which was not rescued by loss of p53. Mtbp+/- mice were not tumor prone. When mice were sensitized for tumor development by p53 heterozygosity, we found that the Mtbp+/-p53+/- mice developed significantly more metastatic tumors (18.2%) as compared to p53+/- mice (2.6%). Results of in vitro migration and invasion assays support the in vivo findings. Downmodulation of Mtbp in osteosarcoma cells derived from p53+/- mice resulted in increased invasiveness, and overexpression of Mtbp in Mtbp+/-p53+/- osteosarcoma cells inhibited invasiveness. These results suggest that MTBP is a metastasis suppressor. These results advance our understanding of the cellular roles of MTBP and raise the possibility that MTBP is a novel therapeutic target for metastasis.

The p53-Mdm2 network in progenitor cell expansion during mouse postnatal development.

Mdm2, an E3 ubiquitin ligase, negatively regulates the tumour suppressor p53. Loss of Mdm2 in mice results in p53-dependent apoptosis and embryonic lethality. This phenotype was rescued by the p53(515C) allele, which encodes an apoptosis-deficient p53R172P protein. However, these mice died within 2 weeks of birth, due to a severe impairment of progenitor cell expansion during postnatal haematopoiesis and cerebellar development, leading to p53-dependent cell cycle arrest. Loss of Mdm2 led to phosphorylation of the p53R172P protein, p53R172P stability and activation of the cell cycle inhibitor p21 in proliferating cells, but not in differentiated cells, in multiple tissue compartments. Proliferating cells of epithelial origin were not affected. The haematopoietic and neural defects were alleviated in mice lacking Mdm2 and containing one p53(515C) and one p53-null allele, but spermatogenesis was arrested. These findings establish a crucial role for the p53-Mdm2 network in regulating proliferation and progenitor expansion in many cell lineages and have important implications for the use of drugs that aim to disrupt the p53-Mdm2 interaction.

Role of prolactin in chloro-S-triazine rat mammary tumorigenesis.

Chloro-S-triazine herbicides [cyanazine (CZ), atrazine (AZ), simazine (SZ)] increase mammary tumors in Crl:CD BR rats but not in F-344 rats or in mice. A nongenotoxic mechanism was investigated since the chloro-S-triazines are negative in short-term tests for genotoxicity. An in vivo battery was used to assess the chloro-S-triazines for estrogenic activity or for their ability to increase prolactin (PRL) levels, both of which play important roles in enhancing mammary gland tumorigenesis in rodents. Ovariectomized (OVX) female rats were treated with AZ, CZ, SZ, or three CZ metabolites for 4 days via intraperitoneal injection. The pattern of responses between the chloro-S-triazines and four controls (estradiol, estriol, haloperidol, reserpine) was compared. For the 6 end-points examined, the responses from rats treated with AZ, CZ, SZ, and the metabolites of CZ most closely matched the responses from the reserpine-treated rats (a PRL rather than estrogenic mechanism). In addition, AZ, CZ, and SZ were tested in several other in vitro models (estrogen/biogenic amine receptor competition assays and a yeast-expressed human estrogen receptor transcription assay) as well as an in vivo 24 h time-course experiment to characterize the CZ-induced increases in PRL levels. AZ, CZ, and SZ are not estrogen receptor (ER) activating compounds based on yeast transactivation and receptor competition data. CZ and AZ demonstrated marginal competition (at mM levels) to the D and alpha2 adrenergic receptors. Ligands to the D2 receptor, but not the alpha2 adrenergic receptor, are known to induce mammary tumors. CZ was also found to produce elevated PRL levels in a time-course similar to that seen with reserpine and haloperidol. Overall, the pattern of responses obtained with the chloro-S-triazines most closely matched the responses observed for reserpine. Taken together, these data suggest chloro-S-triazine-induced mammary tumors in rats are mediated through a PRL mechanism, which is thought to be of low relevance to humans.

Chronic toxicity and oncogenicity bioassay in rats with the chloro-s-triazine herbicide cyanazine.

Cyanazine is a member of the chloro-s-triazine class of herbicides. Other triazine herbicides have been shown to induce mammary-gland tumors in rats, although the response is unique to the Sprague-Dawley strain. Cyanazine is nongenotoxic. The present study was conducted to evaluate the chronic toxicity and oncogenic potential of cyanazine. Groups of 62 male and female rats were fed diets containing cyanazine at concentrations of 1, 5, 25, or 50 ppm for up to 2 yr. Mean body weight and body weight gain of male and female rats of the 25- and 50-ppm groups were significantly reduced over the course of the study. Food consumption and food efficiency were also reduced in these groups. Survival was not adversely affected in the treatment groups compared to controls. A significant increase in the incidence of masses of the inguinal region was noted among female rats of the 50-ppm group. These masses were correlated with a significant increase in the incidence of female rats with mammary-gland adenocarcinomas and carcinosarcomas. The incidence of rats with malignant mammary-gland tumors was elevated in the 5-, 25-, and 50-ppm groups, although the incidence within the 5-ppm group was within historical controls. There were no other toxicologically significant observations with respect to ophthalmological, clinical laboratory, or pathological evaluations. Under the conditions of this study, the no-observed-adverse-effect level was 5 ppm. Research into the mechanism of action suggests these mammary tumors are mediated through a prolactin mechanism that is thought to be of low relevance to humans.

90-day feeding and one-generation reproduction study in Crl:CD BR rats with 17 beta-estradiol.

Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day/one-generation reproduction study with 17 beta-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic toxicity/oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day/one-generation reproduction study was conducted in male and female Crl:CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17 beta-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic/biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P1 generation, dietary administration of 2.5, 10, and 50 ppm 17 beta-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17 beta-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17 beta-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17 beta-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminization of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17 beta-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3% of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17 beta-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17 beta-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F1 generation rats fed 2.5 ppm 17 beta-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17 beta-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17 beta-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F1 generation rats were similar to those observed in the P1 generation rats. (ABSTRACT TRUNCATED)

Weak estrogenic activity from continuous-release pellets.

In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17 beta-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.

An in vivo battery for identifying endocrine modulators that are estrogenic or dopamine regulators.

We have combined several endpoints into a single 5-day in vivo screening procedure to identify estrogenic compounds and dopaminergic modulators, both of which play important roles in enhancing mammary tumorigenesis in rodents. The endpoints evaluated as markers of estrogenicity included increases in uterine fluid and vaginal cornification incidence, serum prolactin levels, uterine weight, uterine epithelial cell height, uterine stromal cell proliferation, and uterine progesterone receptor (PR) number and decreases in uterine estrogen receptor (ER) number. The endpoints evaluated for changes in dopamine regulation included increases in prolactin and decreases in growth hormone levels. The estrogen agonist estradiol (E2) and estriol (E3), the mixed estrogen agonist/ antagonist tamoxifen (TAM), the full antiestrogen ICI-182, 780 (ICI),and the dopamine modulators haloperidol (HAL) and reserpine (RES) were tested using a three-time/day (8-hr intervals) intraperitoneal dosing regimen in sexually mature ovariectomized female Crl:CD BR rats. All compounds were evaluated over a range of concentrations. This in vivo battery was used to evaluate the effects of different classes of endocrine modulators on the selected endpoints. For example, the estrogen receptor agonists E2 and E3 display a unique profile based on changes in the uterotrophic endpoints (estrus conversion, uterine fluid imbibition, increases in uterine weight, and uterine endometrial cell proliferation) where full and partial agonists can be distinguished by the magnitude of these responses. Both the estrogen receptor antagonist ICI and the dopamine modulators HAL and RES lack these uterotrophic responses. Dopamine modulators can be distinguished from estrogen receptor agonists by the profile of increased prolactin levels with no uterotrophic changes. Estrogen receptor antagonists can be distinguished from agonists by comparing their effects on ER, PR, and uterotrophic responses. For instance, the full estrogen receptor antagonist ICI decreased ER (to almost 0) and PR levels, but has no uterotrophic effects, while TAM decreases ER (to almost 0) and increases PR with uterotrophic effects. The most useful endpoints for distinguishing estrogen agonists and dopamine modulators were uterine fluid imbibition, uterine weight, uterine stromal cell proliferation, and serum prolactin levels. In order to distinguish an estrogen agonist from an antagonist, other endpoints, such as receptor levels, are necessary. The advantage of an in vivo screen is that it utilizes a metabolically and physiologically defined system which is especially important with highly integrative system such as the endocrine system. This battery can be used as a screening tool to identify potential endocrine modulators as well as to identify mode of action following adverse findings in toxicology studies. Last, additional endpoints may be added to identify other classes of endocrine modulators.

Hepatic and adrenal changes in rabbits associated with hyperlipidemia caused by a semi-synthetic diet.

Several investigators have reported that feeding a semi-synthetic diet of casein and dextrose to New Zealand White (NZW) rabbits will increase total serum cholesterol concentration, principally through an increase in the beta-lipoprotein fractions, thereby creating a useful model for atherosclerosis research. Although there is evidence to suggest that the dextrose/casein diet alters low-density lipoprotein receptor and bile acid clearance of cholesterol, the underlying mechanism is not completely understood. The effects of the diet on the overall physiology of the rabbit have received little attention. In this study feeding a diet of casein and dextrose of male NZW rabbits for 4 weeks resulted in changes in the serum lipid concentrations. During that time the rabbits fed the dextrose/casein diet gained less weight than did control rabbits. In the test diet rabbits, liver aspartate and alanine transaminase activities were increased from baseline values of 27 +/- 2 U/L and 89 +/- 9 U/L respectively to 112 +/- 21 U/L and 281 +/- 34 U/L respectively, then returned to the high end of the reference range. Necropsy findings included hepatomegaly caused by vacuolar hepatopathy in 19 or 20 experimental rabbits; rabbits fed the control diet had no hepatic lesions. Ultrastructural analysis revealed that enlargement of the liver cells was due to glycogen deposition. Adrenal glands from animals fed the experimental diet had a minimal change in the size of the adrenocortical cells consisting of slight ballooning and rarefaction of the cytoplasm. In a second study the level of dietary fiber was doubled. This resulted in a three-fold increase in lipid concentrations, compared with the fivefold increase in the first study. The liver enzyme activities were increased to the same extent as in the first study. Histologic changes were comparable to those in the first study. The activity of hepatic cholesterol 7alpha-hydroxylase was 3.7 +/- 0.4 pmol/min/mg of protein, compared with the control value of 7.7 +/- 1.1 pmol/min/mg of protein (P < 0.05) in the second study. The improved rate of weight gain and the lesser increase in total serum cholesterol concentration in the second study with increased dietary fiber suggest that two separate activities may be involved. Although the level of dietary fiber may be related to weight gain and total serum cholesterol values, the relation to the decrease in liver transaminase activities in study 1 was probably coincidental. It appears that the dextrose/casein diet causes decreased activity of hepatic cholesterol 7alpha-hydroxylase, which could cause a decrease in the biliary excretion of cholesterol.

Modulation of rat hepatic cytochromes P450 by chronic methapyrilene treatment.

The antihistaminic compound methapyrilene (MP) when chronically administered has been shown to be a rat-specific hepatocarcinogen. To examine the effects of chronic MP treatment on the hepatic microsomal cytochromes P450. Fischer 344 rats were gavaged for 10 weeks (5 days on, 2 days off) with either vehicle or 50, 100, or 150 mg MP/kg body weight. Chronic MP treatment was found to have a significant effect on several microsomal enzymatic activities. Small (17-28%) but significant (P less than 0.05) decreases were observed for total P450 levels and the activities of erythromycin N-demethylase (catalyzed by P450IIIA), N-nitrosodimethylamine demethylase (catalyzed by P450IIE1) and pentoxyresorufin O-dealkylase (catalyzed by P450IIB1). In addition, a relatively large decrease (approximately 80%) was observed for the activity of benzphetamine N-demethylase (representative of P450IIC11) and an induction of about 40% was observed for ethoxyresorufin O-dealkylase (catalyzed by P450IA). The metabolism of testosterone by microsomes isolated from the rats chronically treated with MP indicated that several reactions were compromized. Specifically, testosterone 2 alpha-hydroxylase, indicative of P450IIC11, was reduced greatly (86%), whereas testosterone 6 beta-hydroxylase, reflecting P450IIIA, and testosterone 7 alpha-hydroxylase, indicative of P450IIIA1, were affected only slightly by MP treatment (approximately 25%). Immunoblot analyses of the various microsomal samples were performed to determine if chronic MP treatment had direct effects on the level of expression of the cytochromes P450. Decreases in the levels of P450IIIA, IIE1, and IIC11, determined by immunoblot analyses, closely paralleled those observed for their marker catalytic activities. Further studies will be required to determine the mechanism by which MP affects the levels of the cytochromes P450 (i.e. increased degradation or decreased synthesis).

Catecholamine-synthesizing enzymes and chromogranin proteins in drug-induced proliferative lesions of the rat adrenal medulla.

Both epinephrine (E) and norepinephrine (NE) cells in the rat adrenal medulla are able to proliferate in response to pharmacologic stimulation. However, previous biochemical studies have suggested that drug-induced or spontaneous pheochromocytomas in rats are almost invariably NE-producing. To resolve these apparently conflicting data, immunocytochemical techniques were utilized to establish functional profiles of adrenal medullary lesions classified as pheochromocytoma or nodular hyperplasia in rats treated chronically with a phosphodiesterase inhibitor which induced pheochromocytomas. Sixteen of 17 pheochromocytomas and all hyperplastic nodules stained positively for tyrosine hydroxylase and dopamine beta-hydroxylase, consistent with an ability to produce NE. No lesion of either type stained for phenylethanolamine N-methyltransferase, consistent with an inability to produce epinephrine. Lesions of both types showed variable staining for chromogranin proteins. The findings indicate that qualitative functional differences cannot be used to discriminate hyperplastic nodules from small pheochromocytomas in rats. Some lesions currently classified as hyperplastic nodules might in fact be small pheochromocytomas. Others might represent diffuse hyperplasia within pre-existing islands of NE-cells in a background of hyperplastic epinephrine-cells.

Differential expression of a Mr approximately 90,000 cell surface transferrin receptor-related glycoprotein on murine B16 metastatic melanoma sublines selected for enhanced brain or ovary colonization.

A cell surface Mr approximately 90,000 glycoprotein (gp90) was found in higher amounts on brain-colonizing than on lung-colonizing murine B16 melanoma sublines. The possible role of gp90 in determining the brain-associated metastatic properties of B16 cells was examined by purifying the glycoprotein and studying the effects of anti-gp90 on the growth, adhesion, and organ colonization properties of B16 cells. The specificity of the anti-gp90 was demonstrated in immunoprecipitation studies where a cell surface- or metabolically labeled Mr approximately 90,000 glycoprotein of pI approximately 4 was exclusively found upon two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Immunoprecipitation analysis, enzyme-linked immunosorbent assays, and the lectin-binding properties of gp90 on lectin affinity columns indicated that it is a Mr approximately 180,000 disulfide-linked dimer, probably related to the transferrin receptor. B16 sublines selected for various organ colonization properties differentially expressed gp90, bound 125I-labeled transferrin, and responded differently to purified transferrin in proliferation assays in relation to their metastatic properties (B15b greater than O13 greater than F10 greater than F1). Anti-gp90 immunoglobulin G affected the growth of brain-colonizing B16-B15b more than B16-F1 cells, but had no effect on the adhesion of B16-B15b or -F1 cells to microvessel endothelial cells in vitro, and anti-gp90 immunoglobulin G F(ab')2 had little effect on the brain colonization properties of B16-B15b cells in syngeneic mice. In blocking assays, anti-gp90 inhibited the binding of 125I-labeled transferrin to B16-B15b cells in a dose-dependent manner. The results suggest that the differential growth-stimulating effects of transferrin on highly metastatic B16 melanoma cells may be due to their differential expression of a Mr approximately 90,000 glycoprotein that is related to the transferrin receptor. In organ sites, such as the brain, differential expression of a transferrin-like receptor may allow metastatic cells to respond to low concentrations of growth factors known to be present in certain organs.

An immunocytochemical study of pituitary adenomas and focal hyperplasia in old Sprague-Dawley and Fischer 344 rats.

Spontaneous pituitary tumors occurring in groups of 100 Sprague-Dawley (SD) and 100 Fischer 344 (F344) rats of each sex on a 2-year aging study were characterized by immunocytochemistry. The SD strain had a total of 75 tumors with 10% in males and 65% in females. Tumors immunoreactive for prolactin (PRL) alone were the most common tumor (64%) with the immunonegative tumor being the second most common (17.3%). F344 rats had a total of 62 tumors with 26% in males and 36% in females. The majority of the tumors were reactive for prolactin alone (56.5%) and tumors reactive for both growth hormone (GH) and PRL were the second most common (21%). Most tumors were immunoreactive for only 1 hormone; however, both strains had tumors that expressed multiple hormones in unusual combinations.