Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.     

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.     

TOPOGRAPHY OF DNA SYNTHESIS IN THE MAMMARY GLAND OF THE C3H MOUSE AND ITS CONTROL BY OVARIAN HORMONES: AN AUTORADIOGRAPHIC STUDY

Tritium-labelled thymidine was injected 45 min before sacrifice into virgin female C3H/HeJ mice 7–23 weeks of age, as well as into 10-week-old mice which had been ovariectomized and treated daily with 1 μg of oestradiol-17β and/or 1 mg of progesterone. Autoradiographs were made of squash preparations of the mammary glands, stained by Feulgen's method. The following results were obtained: (1) During normal development of the gland, cells synthesizing DNA are abundant in terminal buds and virtually absent in duct epithelium. Hence ductal growth takes place by the addition of cells produced in the terminal end structures. (2) At 5–6 months, when mammary growth has ceased, a considerable number of cells synthesizing DNA can still be found in alveoli, though not in duct epithelium. Hence the alveolar cells constitute a renewal population in the adult virgin. Because they maintain the potentiality to divide, duct cells are a G₀ population. (3) Ovariectomy results in arrest of DNA synthesis within 3–5 days. Both oestradiol and progesterone restore DNA synthesis in alveoli but only progesterone is able to induce DNA synthesis in duct epithelium, and the differentiation of terminal buds into alveoli. This finding provides an explanation for the resumed proliferation of duct cells in pregnancy. (4) The number of cells engaged in DNA synthesis varies considerably among identical structures within the same gland. This may be due either to synchrony of cell replication and/or to fluctuations of proliferative activity in the gland.     

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

Methylation and amplification of mouse mammary tumor virus DNA in normal, premalignant, and malignant cells of GR/A mice.

The methylation and amplification of mouse mammary tumor virus (MuMTV) proviral DNA was investigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylation pattern was examined with the restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heart was highly methylated. Proviral DNA was somewhat undermethylated in mammary gland cells from virgin and lactating mice and extensively undermethylated in cells from premalignant outgrowths, pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme SacI was used to detect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammary gland cells from virgin mice. Some mammary gland cells from lactating mice appeared to contain additional copies of the endogenous, highly oncogenic GT-MTV-2 provirus. Premalignant outgrowth, pregnancy-dependent tumor, and pregnancy-independent tumor cells contained an average of two to three additional copies per cell of the GT-MTV-2 provirus. Thus, neoplasia in GR/A mice was directly associated with quantized increases in MuMTV proviral DNA undermethylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme analysis suggested that premalignant outgrowths and pregnancy-dependent tumors both consisted largely of heterogenous cell populations, although some evidence of clonal dominance was detected.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

The effect of dietary fat on lipogenesis in mammary gland and liver from lactating and virgin mice

1. Virgin and lactating C(3)H mice maintained on laboratory chow were transferred to a high-fat (15% corn oil) or a fat-free diet 3 days before being killed. 2. The linoleate content of liver, mammary gland and milk was decreased in lactating mice given the fat-free diet but was increased in those fed on the high-fat diet. Changes in linoleate content and mammary gland followed a similar but much less marked trend in virgin animals. 3. Hepatic fatty acid synthesis in lactating and virgin mice fed on the fat-free diet was higher than in corresponding animals fed on either the chow or the high-fat diet. The lipogenic capacity of livers from mice fed on either the chow or the high-fat diet was greater in lactating than in virgin animals. These changes in hepatic lipogenic capacity were accompanied by alterations in the specific activities of certain enzymes involved in fat synthesis. 4. Mammary gland from virgin and lactating animals showed no such adaptation to dietary fat. Results indicate that fatty acid synthesis in neither mammary-gland parenchymal cells nor mammary-gland adipose cells can be influenced by dietary fat in the same way as in the hepatocyte.     

Variation of DNA polymerase activities and DNA synthesis in mouse mammary gland during pregnancy and early lactation

The rate of DNA synthesis and the activity of DNA polymerases and thymidine kinase were measured during the endocrine-regulated cellular growth and differentiation of mouse mammary gland. Using specific assays, the activity of the DNA polymerases, alpha, beta and gamma, was determined in tissue extracts of mammary glands of mice at various stages of pregnancy and early lactation. In addition, extracts of the mammary tissue of virgin, mid-pregnant and early lactating mice were fractionated on sucrose density gradients, and the activity of DNA polymerase alpha and beta was assayed in the gradient fractions. It was demonstrated that the activity of DNA polymerase alpha varied considerably during pregnancy and after parturition, showing peaks on day 12 of pregnancy and days 3-4 of lactation. In pregnancy, there was an apparently parallel correlation between the amount of DNA-polymerase-alpha activity and the rate at which the cells incorporated labelled thymidine into DNA, but the relationship was less clearly expressed during early lactation. The activity of the DNA polymerases, beta and gamma, as well as that of thymidine kinase showed little variation during these periods. Thus, in the developing mammary gland, no correlation was found between DNA synthesis and the activity of the DNA polymerases, beta and gamma, or thymidine kinase.     

Cyclin-dependent kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse mammary preneoplasia.

The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16(INK4a) and p21(Cip1) protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27(Kip1) was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27(Kip1) prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.     

Modulation of alpha2beta1 integrin changes during mammary gland development by beta-oestradiol

In order to study the role of cell-matrix interactions in mammary gland function, temporal changes in alpha2beta1 integrin, the major receptor for collagen and the influence of beta-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of alpha2beta1 integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, alpha2beta1 integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using beta-oestradiol. Supplementing beta-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [35S]methionine into alpha2beta1 integrin associated with the cells. Administration of beta-oestradiol to virgin and lactating glands caused about 1.4-4-fold increase in the level of alpha2 integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of alpha2beta1 integrin occurs. Treatment with beta-oestradiol also resulted in the appearance of alpha2beta1 integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell-ECM interactions through the modulation of integrin receptors for matrix proteins.     

Tissue-specific effects of starvation and refeeding on the disposal of oral [1-14C]triolein in the rat during lactation and on removal of litter.

1. The effects of starvation and refeeding on the disposal of oral [14C]triolein between 14CO2 production and 14C-lipid accumulation in tissues of virgin rats, lactating rats and lactating rats with pups removed were studied. 2. Starvation (24 h) increased 14CO2 production in lactating rats and lactating rats with pups removed to values found in virgin rats. This increase was accompanied by decreases in 14C-lipid accumulation in mammary gland and pups of lactating rats and in white and brown adipose tissue of lactating rats with pups removed. 3. Short-term (2 h) refeeding ad libitum decreased 14CO2 production in lactating rats and lactating rats with pups removed, and restored the 14C-lipid accumulation in mammary glands plus pups and in white and brown adipose tissue respectively 4. Insulin deficiency induced with mannoheptulose inhibited the restoration of 14C-lipid accumulation in white adipose tissue on refeeding of lactating rats with pups removed, but did not prevent the restoration of 14C-lipid accumulation in mammary gland. 5. Changes in the activity of lipoprotein lipase in mammary gland and white adipose tissue paralleled the changes in 14C-lipid accumulation in these tissues. 6. It is concluded that 14C-lipid accumulation in mammary gland may not be affected by changes in plasma insulin concentration and that it is less sensitive to starvation than is lipogenesis or lactose synthesis. This has the advantage that the milk lipid content can still be maintained from hepatic very-low-density lipoprotein for a period after withdrawal of food. The major determinant of the disposal of oral 14C-triolein appears to be the total tissue activity of lipoprotein lipase. When this is high in mammary gland (fed lactating rats) or white adipose tissue (fed lactating rats with pups removed), less triacylglycerol is available for the muscle mass and consequently less is oxidized.     

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.     

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.     

Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation.

Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.     

Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Epithelial cell cycling predicts p53 responsiveness to gamma-irradiation during post-natal mammary gland development

The tumor suppressor gene, TP53, plays a major role in surveillance and repair of radiation-induced DNA damage. In multiple cell types, including mammary epithelial cells, abrogation of p53 (encoded by Trp53) function is associated with increased tumorigenesis. We examined gamma-irradiated BALB/c-Trp53(+/+) and -Trp53(-/-) female mice at five stages of post-natal mammary gland development to determine whether radiation-induced p53 activity is developmentally regulated. Our results show that p53-mediated responses are attenuated in glands from irradiated virgin and lactating mice, as measured by induction of p21/WAF1 (encoded by Cdkn1a) and apoptosis, while irradiated early- and mid-pregnancy glands exhibit robust p53 activity. There is a strong correlation between p53-mediated apoptosis and the degree of cellular proliferation, independent of the level of differentiation. In vivo, proliferation is intimately influenced by steroid hormones. To determine whether steroid hormones directly modulate p53 activity, whole organ cultures of mammary glands were induced to proliferate using estrogen plus progesterone or epidermal growth factor plus transforming growth factor-alpha and p53 responses to gamma-irradiation were measured. Regardless of mitogens used, proliferating mammary epithelial cells show comparable p53 responses to gamma-irradiation, including expression of nuclear p53 and p21/WAF1 and increased levels of apoptosis, compared to non-proliferating irradiated control cultures. Our study suggests that differences in radiation-induced p53 activity during post-natal mammary gland development are influenced by the proliferative state of the gland, and may be mediated indirectly by the mitogenic actions of steroid hormones in vivo.