Insulin-like growth factors 1 and 2 regulate expression of β-casein <Emphasis Type="Italic">in vitro</Emphasis> in mouse mammary epithelial cells

We studied the role of insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) in functional differentiation of HC11 mouse mammary gland cells. It was found that both IGF-1 and IGF-2 activate the expression of milk protein β-casein in the presence of prolactin and hydrocortisone. It was found that β-casein expression is accompanied by cyclin D1 coexpression.     

Effect of transforming growth factor beta 2 on oestrogen metabolism in the MCF-7 breast cancer cell line

Transforming growth factor beta (TGF-beta) is a multifunctional regulator of cellular growth and differentiation in many cell types and has a growth inhibitory effect on mammary epithelial cells. The TGF-beta 2 isoform has been shown to be present in high concentrations in breast cyst fluid and might have a protective role in breast cancer. In addition, oestrogens play an important role in breast cancer development, and oestrone sulphate (E1S) might be the main source of active oestrogens in the breast. The aim of this study was to assess the effect of TGF-beta 2 on oestrogen synthesis in an attempt to understand the mechanism by which TGF-beta 2 may exert a protective effect in breast cancer. In this study, higher concentrations of TGF-beta 2 significantly inhibited the conversion of E1S to oestrone (E1) and the conversion of E1 to the potent oestrogen, oestradiol (E2). TGF-beta 2 did not have any effect on MCF-7 cell growth or on E2 to E1 conversion. In conclusion, TGF-beta 2 might exert a protective role in breast cancer by reducing the amount of active oestrogens present in the breast.     

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland.

Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with beta-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation.     

Stage-dependent effect of TGF-beta1 on chondrogenic differentiation of human embryonic stem cells

Transforming growth factor-beta (TGF-beta) is known to be a potent inducer of stem cell chondrogenic differentiation. Transforming growth factor-beta/activin/nodal-signaling pathway has also been shown to be involved in maintaining the pluripotency of embryonic stem cells (ESCs). In this study, the effect of TGF-beta1 in chondrogenic differentiation of ESCs was examined both with undifferentiated ESCs that bypassed classical embryoid body (EB) formation, and on 5-day EB-derived cells. The effect of TGF-beta1 was compared to cells differentiated in serum-free chondrogenic basal medium without growth factor supplement. Analysis by real-time polymerase chain reaction (PCR), type II collagen enzyme-linked immunosorbent assay, sulfated glycoaminoglycan quantification and fluorescence immunostaining demonstrated substantial chondrogenic differentiation of ESCs regardless of EB formation in the absence of the growth factor. Addition of TGF-beta1 significantly inhibited chondrogenic gene expression and collagen deposition with a more potent effect on the cells that bypassed EB formation. Our study using a TGF-beta/activin/nodal-signaling inhibitor suggested that TGF-beta inhibited early chondrogenic induction but was required at the later stage of differentiation, which was also reflected in the enhancing effect of TGF-beta1 on chondrogenic development at later time points in EB-derived cells. Analysis of the pluripotency markers demonstrated sustained Oct4 and Nanog expression in the presence of TGF-beta1 with Oct4-positive cells detected in subpopulations of the differentiated culture. Our results suggest that TGF-beta1 suppresses ESC chondrogenic induction and the degree of suppression is dependent on the differentiation-stage of the ESC. Transforming growth factor-beta signaling, however, is required for functional chondrogenic development of ESC. Our finding that TGF-beta can sustain an undifferentiated population of human ESCs within the differentiation culture suggests that caution should be exercised when using this growth factor as an ESC chondrogenic inducer and highlights the importance of a selection protocol for chondroprogenitor cells to avoid possible teratoma formation in vivo.     

Vanadate disrupts mammary gland development in whole organ culture.

Protein tyrosine kinases and phosphatases are signaling molecules involved in all aspects of development, including proliferation, differentiation, and apoptosis. How disruption of protein tyrosine phosphatase affects mammary gland development is not entirely clear. We examined the effects of sodium vanadate, which is known to primarily inhibit tyrosine phosphatases, in mouse mammary gland development in whole organ culture. Mammary epithelial differentiation was effectively inhibited by vanadate in a dose-dependent manner as indicated by lack of epithelial alveoli compared to the contralateral non-treated gland controls. Mammary glands in the differentiation medium after four days in the presence of vanadate did not differentiate into alveoli. Instead, they exhibited prominent terminal end buds and lost the distinctive epithelial structures. The inhibitory effect of vanadate on mammary epithelial cell differentiation was irreversible after one day of treatment. Immunohistochemical staining for PCNA (Proliferating Cell Nuclear Antigen) showed that vanadate-treated glands exhibited elevated proliferation signals in the differentiation medium. Expression of beta-casein protein in the vanadate-treated glands decreased dramatically and progressively. Short-term exposure (up to 72 hours) of mammary glands to vanadate resulted in an increase in mammary epithelial cell density and loss of organization of the mammary structures. TUNEL assay of mammary glands with prolonged exposure to vanadate revealed widespread apoptosis. Furthermore, some cells were still proliferating or expressing beta-casein after prolonged exposure to vanadate. Taken together, these data indicate that vanadate treatment blocks mammary epithelial cell differentiation and promotes abnormal proliferation and apoptosis, likely through the inhibition of protein tyrosine phosphatase-mediated signaling.     

Modulation of human IgE synthesis by transforming growth factor-beta.

Exogenous transforming growth factor-beta 2 (TGF-beta 2) markedly inhibits the interleukin 4 (IL4)-stimulated synthesis of human IgE in three models where the B cell co-stimulation signals are contact dependent. This concerns T cell-dependent IgE production by (i) unfractionated peripheral blood mononuclear cells (PMBC) cultured with IL4 and (ii) highly purified B cells cocultured with irradiated EL4 thymoma cells in the presence of IL4 and phorbol myristate acetate, as well as monocyte-dependent IgE production by rigorously T cell-depleted PBMC cultured with IL4 and hydrocortisone. The suppression is not isotype specific. TGF-beta exerts its effect by inhibiting the proliferation of B cells and perhaps also the differentiation of proliferating B cells. However, at a later stage of differentiation, IgE B cells are refractory to the inhibitory effect of TGF-beta, as shown by the slight but significant increase of the spontaneous secretion of IgE by PBMC of atopic patients. This enhancement is due to the suppression of endogenous interferon-gamma production. Most interestingly the synthesis of IgE by highly purified B cells costimulated with IL4 and Epstein-Barr virus is unaffected by TGF-beta. It is concluded that TGF-beta mainly acts by inhibiting IL4-supported B cell proliferation; however, its effects depend upon the B cell costimulation signals that are required together with IL4 for the induction of IgE synthesis.     

Growth inhibitory effects of transforming growth factor-beta 1 in vivo.

Transforming growth factor-beta (TGF-beta) can reversibly inhibit the in vitro proliferation of murine and human haemopoietic progenitors and some of their more developmentally restricted progeny. Using an assay for measuring day 8 and day 11 CFU-S, TGF-beta caused a gradual decline in the number of CFU-S undergoing DNA synthesis so that after 5 days of daily treatment only quiescent cells were found. Release of this growth inhibition was seen within 24 hours post-treatment with recovery of all progenitors to normal levels. Similar inhibitory effects of TGF-beta were seen on the cells of the intestinal epithelium, indicating that TGF-beta is a general stem cell growth inhibitor. These results suggest that TGF-beta can be used as a cytostatic agent to protect normal stem cells in patients being treated with cell cycle-specific cytotoxic agents.     

Regulation of mesangial cell growth by polypeptide mitogens. Inhibitory role of transforming growth factor beta.

Proliferation of mesangial cells is a common histologic abnormality in glomerular diseases. In vivo studies suggest a role for platelets and monocytes-macrophages in mediating glomerular hypercellularity. The authors recently reported that several peptide growth factors stimulate DNA synthesis and growth of human mesangial cells. This article reports that transforming growth factor beta (TGF-beta), a peptide released by inflammatory cells and platelets, inhibits DNA synthesis and growth of human mesangial cells. The stimulatory and inhibitory effects of these mitogens on DNA synthesis and growth was confirmed by autoradiography and cell counting. The inhibitory effect of TGF-beta is not mediated at the receptor level because TGF-beta did not inhibit the binding of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) to mesangial cells. Because peptide growth factors that stimulate DNA synthesis in mesangial cells induce expression of PDGF mRNAs, the effect of TGF-beta on PDGF mRNAs expression induced by peptide growth factors was studied. TGF-beta did not lower the increased levels of PDGF mRNAs caused by EGF or PDGF. These data show that TGF-beta is a potent inhibitor of DNA synthesis and growth of mesangial cells. The mechanism of the inhibitory effect of TGF-beta remains to be determined.     

Transforming growth factor beta: an autocrine regulator of chondrocytes

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.     

Antagonistic effects of IL-4 and TGF-beta1 on Langerhans cell-related antigen expression by human monocytes

In this study, we analyzed the specific effects of transforming growth factor beta (TGF-beta1) and/or IL-4 on monocyte-derived cells. Monocytes were cultured with GM-CSF, GM-CSF/TGF-beta1, GM-CSF/IL-4, or GM-CSF/IL-4/TGF-beta1 before cell morphology, phenotype, and function were assessed. As expected, interleukin-4 is mandatory for monocyte differentiation into potent allostimulatory DC. In its absence, monocyte-derived cells share many phenotypic and functional features with macrophages. However, it is interesting that the cells express E-cadherin, independent of exogenous TGF-beta1, and addition of the cytokine induced CCR6 expression. Most importantly, a subset of monocytes cultured with GM-CSF/TGF-beta1 expresses Langerin, as confirmed by electron microscopy analysis. Langerin engagement with specific monoclonal antibodies induces its internalization and the formation of typical Birbeck granules. Monocytes cultured in GM-CSF/IL-4 did not express the LC markers E-cadherin, CCR6, or Langerin. The simultaneous addition of TGF-beta1 allows most of the cells to express E-cadherin but rarely CCR6 and Langerin. Taken together, the results add further evidence that LC can derive from monocytes and demonstrate an antagonistic effect of IL-4 and TGF-beta1 on monocyte differentiation toward the LC pathway.     

Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells

The effects of soluble mediators and medium supplements commonly used to induce chondrogenic differentiation in different cell culture systems were investigated to define their dose-response profiles and potentially synergistic effects on the chondrogenic differentiation of adipose-derived adult stromal (ADAS) cells. Human ADAS cells were suspended within alginate beads and cultured in basal medium with insulin, transferrin, and selenious acid (ITS+) or fetal bovine serum (FBS) and treated with different doses and combinations of TGF-beta1 (0, 1, and 10 ng/mL) and dexamethasone (0, 10, and 100 nM). Cell growth and chondrogenic differentiation were assessed by measuring DNA content, protein and proteoglycan synthesis rates, and proteoglycan accumulation. The combination of ITS+ and TGF-beta1 significantly increased cell proliferation. Protein synthesis rates were increased by TGF-beta1 and dexamethasone in the presence of ITS+ or FBS. While TGF-beta1 significantly increased proteoglycan synthesis and accumulation by 1.5- to 2-fold in the presence of FBS, such effects were suppressed by dexamethasone. In summary, the combination of TGF-beta1 and ITS+ stimulated cell growth and synthesis of proteins and proteoglycans by human ADAS cells. The addition of dexamethasone appeared to amplify protein synthesis but had suppressive effects on proteoglycan synthesis and accumulation.     

Role of TGF-β and the tumor microenvironment during mammary tumorigenesis

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to inhibit mammary tumorigenesis by directly inducing mammary epithelial cells (MECs) to undergo cell cycle arrest or apoptosis, and to secrete a variety of cytokines, growth factors, and extracellular matrix proteins that maintain cell and tissue homeostasis. Genetic and epigenetic events that transpire during mammary tumorigenesis typically inactivate the tumor suppressing activities of TGF-beta and ultimately confer this cytokine with tumor promoting activities, including the ability to stimulate breast cancer invasion, metastasis, angiogenesis, and evasion from the immune system. This dramatic conversion in TGF-beta function is known as the "TGF-beta paradox" and reflects a variety of dynamic alterations that occur not only within the developing mammary carcinoma, but also within the cellular and structural composition of its accompanying tumor microenvironment. Recent studies have begun to elucidate the critical importance of mammary tumor microenvironments in manifesting the TGF-beta paradox and influencing the response of developing mammary carcinomas to TGF-beta. Here we highlight recent findings demonstrating the essential function of tumor microenvironments in regulating the oncogenic activities of TGF-beta and its stimulation of metastatic progression during mammary tumorigenesis.     

Role of compensatory growth in lactation: a stair-step nutrient regimen modulates differentiation and lactation of bovine mammary gland

Twenty Holstein heifers were assigned to either a control or test (stair-step compensatory; food restriction, followed by refeeding) growth group. The stair-step growth model was designed to induce distinctive compensatory allometric mammary development during three different hormonal states, coinciding with prepubertal, pubertal, and late gestational stages. Mammary tissues obtained by biopsy from pregnant and lactating cows were used for acini culture and chemical composition analysis. Test mammary tissues from late gestation heifers contained less (P = .067) fat than control counterparts (731 vs. 628 mg/g). DNA, RNA, and protein contents in test mammary tissue from late gestation cows were higher (P = .001 to .088) compared to control tissue. Milk protein secretion of test acini in culture was increased more than 20% over that of the control acini. Lactating mammary acinar cells in culture from test cows exhibited a 14% increase in amino acid uptake over that of the control. RNA dot-blot hybridization analysis revealed that alpha s1- and beta-casein mRNA accumulation in acini from test tissue was increased (P = .027 to .042) as much as 40-50%. During both food restriction and refeeding, concentrations of plasma growth hormone were elevated. Food restriction decreased levels of plasma insulin, whereas levels of insulin were elevated during refeeding. The long-term influence of compensatory growth upon subsequent lactation performance was also evaluated. Milk production data derived from previous two sequential growth trials showed that cows from test groups produced approximately 10% more (P = .001) milk compared to control counterparts. Results reinforce our postulation that compensatory growth induced by nutritional modulation regulates the differentiation and functional activity of the mammary gland.     

Effect of porcine fetal enamel matrix derivative on chondrocyte proliferation,differentiation, and local factor production is dependent on cell maturation state

Recent studies have shown that porcine fetal enamel matrix derivative (EMD) can enhance the osteoinductive ability of demineralized freeze-dried bone allograft (DFDBA) in a nude mouse muscle implantation model. This suggests that one or more components of EMD can regulate the process of endochondral ossification initiated by DFDBA. To substantiate this hypothesis, in the current study, chondrocytes in the endochondral pathway at two stages of maturation were tested for their response to EMD. Chondrocytes were isolated from the resting zone and growth zone (prehypertrophic and upper hypertrophic cell zones) of the costochondral growth plate cartilage of adolescent rats. The results showed that the relatively immature resting zone cells responded to EMD with an increase in proliferation and a decrease in differentiation as measured by alkaline-phosphatase-specific activity. In addition, EMD stimulated a fivefold increase in PGE(2) production, but was without effect on collagen synthesis, proteoglycan sulfation, and TGF-beta(1) production. The more mature growth zone cells also responded to EMD with increased proliferation, but alkaline-phosphatase-specific activity was unchanged, and there was only a modest increase in PGE(2) production. In contrast to resting zone cells, growth zone cells exhibited a decrease in collagen synthesis, proteoglycan sulfation, and TGF-beta(1) production. These observations indicate that EMD has prominent effects on cells in the endochondral pathway. In particular, EMD stimulates the production of more cells, but inhibits their maturation. This would increase the pool of cells available for subsequent differentiation in response to other factors.     

Effects of transforming growth factor-beta, transforming growth factor-alpha, and other growth factors on renal proximal tubule cells

Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.     

Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents.

Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture.     

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.     

Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice

Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.