Reduction of estrogen-induced transformation of mouse mammary epithelial cells by N-acetylcysteine

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-Catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-Acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well tolerated in clinical studies, it may be a promising candidate for breast cancer prevention.     

Differential transformation of mammary epithelial cells by Wnt genes.

The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent.     

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.     

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.     

The mouse Wnt-1 gene can act via a paracrine mechanism in transformation of mammary epithelial cells.

The mouse Wnt-1 gene plays an essential role in fetal brain development and can contribute to tumorigenesis when activated aberrantly in the mammary gland. The gene encodes secretory glycoproteins associated with the extracellular or pericellular matrix, and it has been proposed that Wnt-1, as well as its Drosophila homolog wingless, may function in intercellular signalling. We show here that fibroblasts expressing Wnt-1 protein, although not transformed themselves, are able to elicit morphological transformation of neighboring C57MG mammary epithelial cells in coculture experiments. Heparin inhibits this effect, possibly by displacing Wnt-1 protein from its normal site of action. Our results indicate that the Wnt-1 gene can act via a paracrine mechanism in cell culture and strongly support the notion that in vivo the gene may function in cell-to-cell communication.     

Induction of lactoferrin gene expression by innate immune stimuli in mouse mammary epithelial HC-11 cells

Lactoferrin (LF) is a multifunctional protein. While its functions and mechanism of actions are actively being investigated, the cellular signals that regulate LF expression have not been as explored. We have previously demonstrated that LF is upregulated by estrogen in the reproductive system. In this study, we show that the expression of LF was stimulated by bacterial lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) in normal mouse mammalian HC-11 cells. When cells were exposed to either LPS or dsRNA, the mRNA and protein of LF were increased in a dose- and time-dependent manner, yet the kinetics of LF induction by dsRNA or LPS were different. The LPS and dsRNA-induced LF was mainly released into the culture medium where it blocked TNF-alpha production in exposed cells. We explored the mechanisms of LF induction by LPS and dsRNA using specific inhibitors and found that the induction could be attenuated by inhibitors to PKC, NF-kappaB, p38 and JNK, but not by an inhibitor to PKA. Interestingly, ERK inhibitor was effective against dsRNA but not against LPS induction of LF. These data suggest that LF was induced by LPS and dsRNA through PKC, NF-kappaB and MAPK pathways which in turn play an inhibitory role in the continuation of innate inflammation.     

Persistent estrogen responsiveness of ras oncogene-transformed mouse mammary epithelial cells.

Ovarian steroids are associated with the proliferation of normal as well as tumorigenically transformed mammary epithelial cells. The experiments performed in this study were designed to establish that (1) tumorigenic transformation induced by the ras oncogene is associated with alterations in estradiol biotransformation, (2) altered endocrine responsiveness persists in the fully transformed tumor cell phenotype and (3) specific perturbations induced by the ras oncogene can be experimentally downregulated. The ras transfectant pH06T and the tumor-derived T1/Pr1 cells exhibited 3- and 43-fold increases, respectively, in C-16 alpha hydroxylation of estradiol relative to the parental mouse mammary epithelial cells (P less than 0.0001). At the cellular level, this alteration corresponded with approximately 90-fold increase in the anchorage-independent growth of T1/Pr1 cells (P less than 0.0001). Estrogen responsiveness of T1/Pr1 cells was demonstrated by their suppression of growth in phenol red-free and/or tamoxifen-supplemented medium and by the reversal of antiproliferative effect of tamoxifen by phenol red and estradiol. Indole-3-carbinol, a naturally occurring tumor suppressive agent, was able to upregulate C-2 hydroxylation at the expense of C-16 alpha hydroxylation of estradiol. Treatment of T1/Pr1 cells with indole-3-carbinol resulted in a substantial decrease in anchorage-independent growth.     

To grow mouse mammary epithelial cells in culture

Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures.     

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.     

Cul5 mediates taurine-stimulated mTOR mRNA expression and proliferation of mouse mammary epithelial cells

Cullin5 (Cul5) protein can regulate multiple signaling pathways; however, it is still largely unknown the role and molecule mechanism of Cul5 in regulation of the mTOR signaling. In this study, we determined the effect of Cul5 on the proliferation of HC11 cells, a mouse mammary epithelial cell line, and explored the corresponding molecular mechanism. We found that Cul5 was highly expressed in mammary gland tissues in the lactation stage compared with that in puberty and involution. Using gene knockdown and activation methods, we showed that Cul5 promoted proliferation of HC11 cells, mRNA expression and protein phosphorylation of mTOR. Taurine (Tau) affected Cul5 mRNA and protein levels in a dose-dependent manner. Cul5 localized to the nucleus and knockdown of Cul5 almost totally blocked the stimulation of Tau on mTOR mRNA expression and protein phosphorylation. PI3K inhibition almost totally abolished the stimulation of Tau on Cul5 expression. In summary, our data uncover that Cul5 is a positive regulator of proliferation of HC11 cells, and mediates the stimulation of Tau on mRNA expression and subsequent protein phosphorylation of mTOR. Our data lay a new theoretical foundation for regulating mammary cell proliferation and promoting milk yield.

     

Trichostatin A inhibits beta-casein expression in mammary epithelial cells.

Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.     

Mechanism of the decrease in hexose transport by mouse mammary epithelial cells caused by fasting.

The basal carrier-mediated uptake of 0.5 mM-3-O-methylglucose by mammary epithelial cells from lactating mice was calculated to be 227 +/- 9 pmol/min per microgram of DNA (mean +/- S.E.M., n = 11). Fasting the mice for 16 h overnight resulted in a decrease in this rate to 65 +/- 4 pmol/min per microgram of DNA (n = 10). Refeeding the fasted mouse for 3 h before isolation of the cells restored the transport activity to 230 +/- 12 pmol/min per microgram of DNA (n = 12). The Vmax. for equilibrium exchange entry of 3-O-methylglucose by intact cells was decreased from 6.6 +/- 0.4 to 0.9 +/- 0.2 nmol/min per microgram of DNA (mean +/- S.E.M., n = 3) by fasting. The number of D-glucose-inhibitable cytochalasin-B-binding sites in a plasma-membrane-enriched fraction of the cells was also decreased from 5.7 +/- 1.5 to 1.7 +/- 0.1 pmol/mg of membrane protein (mean +/- S.E.M., n = 3). Again, refeeding the fasted mouse for 3 h reversed both these effects. These results are consistent with a decrease in the number of functional glucose carriers in the plasma membrane of the mammary epithelial cells. Since the restoration of transporter activity after refeeding does not appear to require the synthesis of new protein, the effect of fasting probably involves not a loss of transporters, but a change in their orientation within the plasma membrane or a redistribution within the cell.     

Regulation of clusterin expression in mammary epithelial cells

Mammary epithelial cells undergo changes in growth, invasion, differentiation, and dedifferentiation throughout much of adult hood, and most strikingly during pregnancy, lactation, and involution. Clusterin is a multifunctional glycoprotein that is involved in the differentiation and morphogenesis of epithelia, and that is important in the regulation of postnatal mammary gland development. However, the mechanisms that regulate clusterin expression are still poorly understood. Here, we show that clusterin is up-regulated twice during mouse mammary gland development, a first time at the end of pregnancy and a second time at the beginning of the involution. These points of clusterin up-regulation coincide with the dramatic phenotypic and functional changes occurring in the mammary gland. Using cell culture conditions that resemble the regulatory microenvironment in vivo, we determined that the factors responsible for the first up-regulation of clusterin levels can include the extracellular matrix component, laminin, and the lactogenic hormones, prolactin and hydrocortisone. On the other hand, the second and most dramatic up-regulation of clusterin can be due to the potent induction by TGF-beta1, and this up-regulation by TGF-beta1 is dependent on beta1 integrin ligand-binding activity. Moreover, the level of expression of beta-casein, a marker of mammary epithelial cell differentiation, was decreased upon treatment of cells with clusterin siRNA. Overall, these findings reveal several novel pathways for the regulation of clusterin expression during mammary gland development, and suggest that clusterin is a morphogenic factor that plays a key role during differentiation.     

Regulation of the mouse mammary tumor virus receptor by phosphorylation and internalization in mammary epithelial cells

The mouse mammary tumor virus enters mammary epithelial cells via a plasma membrane protein that binds to a viral envelope glycoprotein, gp52. In intact cells, this gp52 receptor can be phosphorylated by activators of protein kinase A and protein kinase C (PKC), but this modification does not occur in response to epidermal growth factor, whose receptor is a tyrosine kinase, or to gp52. Phosphorylation of the gp52 receptor rapidly leads to internalization and gradual loss of binding activity. Both the phosphorylation and the intrnalization induced by PKC are abolished by prior downregulation of this kinase. Although the physiological function of the gp52 receptor is unknown, its binding to gp52 can stimulate several biological activities, including amino acid accumulation. Receptor processingimpairs this gp52-induced amino acid uptake, as well as viral infection, by depleting the binding protein at the cell surface. In contrast, PKC augments insulin-induced amino acid transport, and PKC downregulation abolishes the action of insulin, suggesting that insulin and gp52 utlize partially separate pathways leading to amino acid transport. These data further suggest that PKC may be involved in this insulin-stimulated activity. © 1994 Wiley-Liss, Inc.     

Taurine attenuates lipopolysaccharide-induced disfunction in mouse mammary epithelial cells

The intent of this study was to evaluate the active defense reaction of mouse mammary epithelial cells and the cytoprotective and anti-inflammatory properties of taurine to lipopolysaccharide (LPS)-induced disfunction in mouse mammary epithelial cells. (1) Primary cultured mouse mammary epithelial cells were stimulated with LPS for 24 h (final concentration=0, 5, 10, 20 μg/mL). Western blotting demonstrated a significant decrease in the secretion of β-casein in the 20 μg/mL LPS treatment group (P<0.05), while nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), lactoferrin (LF) and N-acetyl-β-D-glucosaminidase (NAGase) were all significantly increased following LPS treatment (P<0.01). Furthermore, cell survival was significantly inhibited after treatment with 20 μg/mL LPS; however, neither 5 μg/mL nor 10 μg/mL LPS had any effect on cell survival. Therefore, a level of 10 μg/mL LPS was selected to test the protective effect of taurine on mouse mammary epithelial cells. (2) Primary cultured mouse mammary epithelial cells were treated with 0, 5, 15 or 45 mmol/L taurine for 3 h, followed by 10 μg/mL LPS for 24 h. Taurine significantly attenuated the LPS-induced increase in NAGase activity, NO concentrations and the level of TNF-α, IL-1β, IL-6 and LF. Taurine at 45 mmol/L markedly increased β-casein secretion in response to LPS-induced disfunction. This study demonstrated that the addition of taurine to a culture medium significantly inhibited the LPS-induced release of inflammatory factors and increased β-casein secretion from mammary epithelial cells, thereby providing a possible explanation for the protective effect proposed for taurine in the prevention of LPS-induced disfunction in mammary epithelial cells.     

Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross- linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. alpha-Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.