PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool

Wenjing Li; Brian J. Ferguson; Walid T. Khaled; Maxine Tevendale; John Stingl; Valeria Poli; Tina Rich; Paolo Salomoni; Christine J. Watson

doi:10.1073/pnas.0807640106

Edited by Mina Bissell, Lawrence Berkeley National Laboratory, University of California, Berkeley, and accepted by the Editorial Board February 10, 2009
↵¹W.L. and B.J.F. contributed equally to this work. (received for review August 8, 2008)

Abstract

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

Keywords:

The promyelocytic leukemia protein (PML) is a tumor and growth suppressor and pleiotropic regulator of multiple cellular processes including cell death, differentiation, and stress response pathways (1–3). PML resides in large macromolecular protein complexes, known as PML nuclear domains PML-NDs (PML bodies/ND10/POD), of which PML is an essential component (2). The role of PML-NDs is enigmatic (1, 3), but their presence is vital for the correct function of PML protein as evidenced by the role of PML in acute promyelocytic leukemia (APL) and by murine models of PML loss. The majority of acute promyelocytic leukemia (APL) cases involve the t (15, 17) chromosomal translocation that results in a genetic fusion of the PML gene with that of the retinoic acid receptor-α (RARα) (4). The resulting fusion protein, PML-RARα, acts as dominant negative mutant of both proteins, leading to loss of PML-NDs, a block in myeloid cell differentiation and subsequent leukemogenesis (4, 5). Loss of PML in murine models leads to a resistance to apoptosis, increased cellular growth rates, and a higher sensitivity to a range of tumor-inducing treatments and mutations, underlining the importance of PML in a host of physiological processes (6, 7). However, the PML null mice are conspicuous for their lack of developmental phenotypes other than a mild general anemia (7), although recent data have pointed to a role for PML in preserving tissue stem cell populations (8).

PML expression has been widely studied in both normal and neoplastic tissues (9–15). Expression is widespread in normal tissues; however, in general (and outside of the stem cell compartment), high levels of PML are associated with an inflammatory cytokine environment (9) and regions of high cellular proliferation (9), although some evidence disputes this (11). PML is also down-regulated during differentiation in several tissues such as the myeloid lineage and several epithelia (8, 11, 12, 16). PML expression is disregulated in many types of neoplasia (10, 13), consistent with its roles in regulation of cell growth, fate, and differentiation. Despite the extensive studies of PML protein expression, there exists little knowledge of the molecular mechanisms that underlie its transcriptional regulation. Pml is, however, a well-characterized interferon (IFN) response gene (17, 18), with promoter elements [interferon-stimulated response element (ISRE) and interferon γ-activated (GAS) sites] that function as binding sites for the signal transducer and activator of transcription (Stat) family of transcriptional regulators (19). Hence the up-regulation of Pml in an inflammatory cytokine environment can be explained by classical activation of the Janus kinase (JAK)/Stat signaling pathway by interferons. In addition, the inhibitory action of PML on Stat1α activity (activated by IFN-γ) in murine embryonic fibroblasts (MEFs) reveals a complex pattern of PML induction and negative feedback (20).

The Stat proteins act as key transcription factors in response to cytokine signaling, thereby modulating many survival, growth, and differentiation pathways. Stat activities are known to play a major role in the development of the mammary gland (21), a tissue that provides an excellent model of differentiation, death, and remodeling. The mammary gland epithelium is set in an adipocyte-rich stroma (known as the fat pad) and undergoes extensive proliferation and differentiation in response to steroid hormones. During puberty, ductal epithelial cells (luminal and myoepithelial) proliferate to form a branched ductal network. At the onset of pregnancy, the epithelial cells begin to proliferate again in response to progesterone, resulting in tertiary branching and formation of lobuloalveolar structures that are the site of milk production. Finally, after weaning, the mammary gland undergoes a period of extensive cell death and remodeling known as involution. These distinct phases of development are tightly controlled by a delicate balance of hormones and cytokines. Furthermore, it is the hierarchy of expression of the Stat proteins that helps to govern the responses of the mammary gland to such developmental signals (21, 22). Hence, Stat6 loss is associated with a delay in alveolar development during pregnancy (23). Prolactin-mediated Stat5 activity stimulates functional lobuloalveolar development during late pregnancy and milk production during lactation (24), whereas leukemia inhibitory factor (LIF) induces Stat3 activity at the onset of involution to initiate the death and re-modeling processes (25). In this study it is shown that interplay between PML and Stats 3, 5, and 6 helps to control the normal development of the mammary gland during a pregnancy/lactation/involution cycle. Furthermore, in non-pregnant animals, PML loss results in reduced ductal diameter and is, surprisingly, a regulator of branching morphogenesis. Finally, it is demonstrated that PML loss is also associated with an imbalance in the populations of progenitor cells that contribute to the different luminal epithelial lineages in the gland.

Results

PML Is Transcriptionally Regulated During Mammary Gland Development.

The expression of PML protein was found to be tightly regulated, at both the transcript and protein level, during the distinct phases of mammary gland development (MGD) (Fig. 1). The virgin gland was found to contain relatively high levels of PML protein, as analyzed by immunoblotting of whole-gland preparations (Fig. 1A) and immunohistochemistry of murine tissue sections (Fig. 1B). Expression was found in all cell types but was especially prominent in the epithelium (data not shown). However, during gestation and lactation the amount of PML protein declined to undetectable levels; and after 10 days of lactation (10L) PML-NDs were no longer observed in the epithelium (Fig. 1B), although they remained present in other cell types. As immunoblotting was carried out on whole glands that contained a mixture of epithelia, fibroblasts, and adipocytes, the level of PML was compared with that of the epithelial marker E-cadherin (Fig. 1A). From these data, it is clear that the levels of PML isoforms 1 and 2 are high in the mammary epithelium of virgin mice when PML-NDs are large and numerous (Fig. 1B), and decline during lactation, with no PML-NDs detectable at 10L. Remarkably, during the initial stages of involution, the level of PML protein increases as evidenced by the re-forming of small microbodies of PML (Fig. 1B) that eventually nucleate many large PML-NDs at later time-points.

Fig. 1.

PML expression during mammary gland development. (A) Murine mammary glands from virgin (V), gestation (G), lactation (L), and involution (I) time points (in days) as indicated were probed for PML protein expression by immunoblotting. PML appears as two bands corresponding to the two alternatively spliced isoforms labeled 1 and 2 respectively. The expression levels of PML isoforms 1 and 2 relative to E-Cadherin (E-Cad) are shown in the lower panel. (B) Nuclear PML-NDs (red) were visualized by immunohistochemistry, with DAPI counterstain, on mammary gland sections from various stages of development. Scale bars, 5 μm. (C) RNA levels of PML isoforms 1 and 2 were measured by quantitative RT-PCR and are expressed relative to the level of cyclophilin mRNA. (D) KIM2 cells were differentiated with prolactin and dexamethasone for the number of days indicated and probed for PML expression. The transferrin receptor (TR) was used as a loading control.

Examination of the levels of PML mRNAs in the same tissues indicated transcriptional regulation of PML in keeping with the pattern of protein changes (Fig. 1C). Transcripts corresponding to both known splice variants of murine PML were tested and found to be similarly regulated although the differential expression of either transcript was not as marked as that seen at the protein level. This suggests that at the protein level, the two PML isoforms may be differentially regulated. Protein levels of the smaller isoform (isoform 2, lower band in Fig. 1A) were higher in the virgin gland, whereas the larger isoform (isoform 1, upper band in Fig. 1A) is most abundant in early gestation.

PML loss has been associated with the aberrant differentiation of specific cell types, particularly those of the hematopoietic and epithelial compartments (8, 11, 12, 16). The suppression of PML during the gestation and lactation stages of mammary gland development was therefore postulated to be associated with the differentiation status of the mammary epithelium. To test this, the conditionally immortal murine mammary epithelial cell line KIM-2 was tested for PML expression during a time-course of treatment with prolactin and dexamethasone. This lactogenic hormone mixture results in the differentiation of these cells in a manner that mimics the in vivo progression of the mammary epithelium from virgin through gestation to lactation (26); and, indeed, PML expression was found to reduce over the course of this treatment (Fig. 1D). Notably, expression of PML isoform 2 was almost completely lost by day 10 of the KIM-2 differentiation time course, a stage that approximates mid-lactation, consistent with the in vivo results.

PML Expression Is Regulated by Multiple Stat Activities.

The Stat proteins are key regulators of mammary gland development, and several members of this family of transcription factors have been implicated in multiple aspects of this process (23–25). The presence of GAS and ISRE sites in the PML promoter suggested that the Stat proteins may be responsible for the regulation of PML expression in vivo. To provide clues as to which Stats may be responsible for such regulation, Stats were focused on that have been shown previously, using gene deletion studies, to have critical roles in normal mammary gland development during a pregnancy/lactation/involution cycle. Stat6 is important in early gestation in promoting proliferation and development of lobuloalveolar cells (23), whereas Stat5 is an essential mediator of prolactin signaling and is required for functional differentiation of lobuloalveoli and for milk secretion (24). Stat3 is specifically activated at high levels at the onset of postlactational regression (involution) when it is critical for this process (27). The unique activation profile of these individual Stats [supporting information (SI) Fig. S1] suggests that the balance of multiple Stat activities may affect the level of PML expression during MGD.

Activation of Stat3 or Stat6 Results in Decreased PML Expression.

The role of Stat proteins in regulating PML expression was analyzed using both in vivo and in vitro models. Treatment of the mammary epithelial cell line EpH4 with oncostatin M (OSM), a direct and strong inducer of Stat3 activity, down-regulated PML expression in vitro within 24 h, as measured by immunoblot and immunofluorescence (Fig. 2A). Furthermore treatment with prolactin activated Stat5 and mediated suppression of PML by 8 h of treatment (Fig. 2B), whereas IL-13, which activates Stat6, down-regulated PML expression in as little as 2 hours (Fig. 2C). Similar results were obtained in vivo using mice deficient for Stat3 or Stat6. Microarray analysis of mammary glands at 24 hours involution in which Stat3 had been conditionally deleted showed that the Pml gene was up-regulated in comparison to wild-type glands. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted from the same glands confirmed that Pml transcription was increased up to 8-fold in the Stat3 null glands (Fig. 2D). This transcriptional increase was reflected by an increase in the number and intensity of PML-NDs in the epithelium (Fig. 2E) and by an increase in PML protein level (Fig. 2F). Similar results were obtained in studying the interplay between PML and Stat6. At day 5 of gestation in Stat6 null mouse mammary glands, PML levels were found to be up-regulated with respect to the wild-type glands, and the number and intensity of PML-NDs in the epithelium was increased (Fig. S2). It is clear, then, that the balance of Stat activities in vivo results in the overall regulation of PML protein levels in the mammary epithelium.

Fig. 2.

Stat activity negatively regulates PML protein expression in vitro and in vivo. EpH4 cells were treated with (A) oncostatin M, (B) prolactin, or (C) IL-13 for the indicated times and were probed for active Stat 3, 5, and 6, respectively, and PML. A representative fluorescence micrograph of PML immunostaining after oncostatin M treatment is shown (A, lower panel), highlighting the reduction in number and intensity of PML-NDs after treatment. Scale bars, 5 μm. (D) Glands from wild-type (Stat3^flox/flox) and Stat3 conditional knockout (BLG-Cre/Stat3^flox/flox) mice at 24 hours' involution were analyzed for PML mRNA expression by quantitative RT-PCR and for protein expression by (E) immunofluorescence (scale bars, 5 μm) and (F) immunoblotting (left panel). Immunoblots were analyzed by densitometry (right panel) to indicate the increased level of PML expression in Stat3 null mice (n = 3, ***P < 0.01).

On the basis of these data, we hypothesized that PML may influence MGD. Glands from Pml^−/− mice at 15 days gestation were analyzed and found to contain reduced levels of secreted β-casein and the phosphorylated form of Stat5 (Fig. 3 A and B), two functional markers of alveolar development. Importantly, this was associated also with a reduction in the level of pStat6 (Fig. 3B), suggesting that levels of PML are exquisitely controlled to ensure normal mammary gland development and that overexpression of PML (as seen in Stat6^−/− glands) or lack of expression (as in the Pml^−/− glands) delays differentiation of mammary alveolar structures.

Fig. 3.

PML influences Stat activation and MGD. (A) Pml^+/− and Pml^−/− mammary glands from 15-day-gestation mice were analyzed for β-casein expression (red) by immunofluorescence (scale bars, 10 μm). (B) Glands from Pml^+/− and Pml^−/− mice at 15 days gestation were analyzed for Stat 5 and Stat 6 activation by immunoblotting.

PML Regulates Ductal and Branching Morphogenesis in Vivo.

Considering that mammary ductal morphogenesis occurs primarily during adolescent development and that PML is a known growth suppressor (7), glands from 6-week-old virgin Pml^−/− mice were analyzed for their architectural characteristics and compared with those of heterozygous littermates. Whole-gland mounts and hematoxylin and eosin (H&E)-stained sections from mice lacking PML were found to have significantly smaller ducts with reduced numbers of branches per duct (Fig. 4A, Fig. S3). Quantitative analysis of these data indicated that lumen size in ducts from Pml^−/− mammary glands was reduced by an average of 65%, whereas the number of branch points per duct was reduced by 40% (Fig. 4B). These data uncover a surprising role for the PML protein in the regulation of in vivo ductal morphogenesis. Further analysis of these glands by immunohistochemical staining for markers of the ducto-luminal (cytokeratin 18; CK18) and ducto-basal (smooth muscle actin; SMA) epithelial cells showed that, although the ducts were globally smaller, the cellular organization remained normal, and luminal cells are correctly polarized as shown by the luminal surface localization of aquaporin 5 (Fig. 4A).

Fig. 4.

PML loss affects mammary gland morphogenesis. (A) Individual ducts from H&E-stained and immunostained sections of Pml^+/− and Pml^−/− mammary glands from 6-week-old virgin mice are shown, highlighting the smaller size but retained architecture of Pml^−/− ducts. Immunostains are for cytokeratin 18 (CK18), smooth muscle actin (SMA), and aquaporin 5 (AQP5). (B) H&E-stained sections and whole mounts were quantified in terms of duct lumen cross-sectional area (n = 30, ***P < 0.01) and number of branch points per duct (n = 10, ***P < 0.01), respectively (lower panels).

PML Influences Mammary Epithelial Differentiation by Altering Progenitor Populations.

Not only was ductal organization unaffected by PML depletion, but equally no alterations could be found in the amount of proliferation or cell death in virgin Pml^−/− mammary glands (Fig. S4). This led us to suspect that PML may affect the differentiation of certain populations of cells within the gland. Recently, mammary gland differentiation pathways have begun to be delineated with the identification of discrete populations of progenitor cells that are capable of growing in vitro, repopulating cleared mammary fat pads, and specifically differentiating into subsets of functionally different epithelial cells (28, 29). Luminal epithelial cells can be divided into differentiated (CD24^hi CD49b⁻) and progenitor (CD24^hi CD49b⁺) compartments based on their ability to form colonies in vitro. The progenitor compartment can be subdivided further based on Sca1 staining into Sca1⁻ (ERα⁻ luminal progenitors) and Sca1⁺ (ERα⁺ luminal progenitors) populations (Fig. S5). In this study, the Sca1⁻ and Sca1⁺ progenitor populations from the Pml^+/− and Pml^−/− mammary glands were analyzed. The Sca1⁻ luminal progenitors were significantly reduced in Pml^−/− mice (20 ± 1.9%) compared with Pml^+/− mice (53 ± 4.6%) (Fig. 5A). In contrast, there was an increase in the number of Sca1⁺ cells in Pml^−/− mice (57 ± 14%) compared with Pml^+/− mice (30 ± 2%) (Fig. 5A). These populations were sorted and analyzed for their cloning efficiency; surprisingly, the cloning efficiency of unsorted (total) epithelial cells was significantly greater in Pml^−/− compared to Pml^+/− mice (Fig. 5B). Epithelia from heterozygotes generated 4.4 (±0.35) colonies per 100 cells, whereas the knock-out animal generated 6.6 (±0.2) colonies (P < 0.02). This increase is most likely caused by the increase in the cloning efficiency of the Sca1⁺ luminal progenitors (Fig. 5B). Consistent with these results, immunostaining for estrogen receptor (ERα) revealed a marked increase in the number of positive cells in Pml^−/− glands compared to Pml^+/− (Fig. 5C and 5D). Interestingly, although there is a reduction in the number of Sca1⁻ luminal progenitors, PML deficiency did not affect the cloning efficiency of this reduced population (Fig. 5B). RT-PCR analysis of RNA from purified subpopulations of cells revealed that expression of the Stat6 target GATA-3, which has been shown to be important in establishment and maintenance of the luminal lineage (29), is suppressed in the Sca1⁻ progenitor population from Pml^−/− glands compared to controls (Fig. 5E). Interestingly, NOTCH-1, also a regulator of GATA-3 (30), was expressed at very low levels in the Sca1⁻ cells, suggesting that Stat6 is the primary regulator of GATA-3 in this population. These data indicate that PML regulates both the ability of mammary progenitors to proliferate as well as the balance of specific progenitor populations. Loss of PML therefore affects mammary differentiation by skewing the relative populations of specific epithelial lineages.

Fig. 5.

PML loss influences mammary epithelial progenitor populations (A) Numbers of Sca1⁺ and Sca1⁻ luminal progenitors shown as a percentage of live luminal cells (CD24^hi) assayed by flow cytometry from 14-week-old Pml^+/− and Pml^−/− virgin mammary glands highlighting the reduction in Sca1⁻ and increase in Sca1⁺ populations in KO tissue (n = 2, ***P < 0.01, **P < 0.02). (B) Cloning efficiency (per hundred cells) of FACS sorted Sca1⁺, Sca1⁻ luminal progenitors and total epithelial cells from 14-week-old Pml^+/− and Pml^−/− mice (n = 2; ***P < 0.01, **P < 0.02). (C) Immunohistochemistry for ERα (red) and E-Cadherin (green) on mammary gland sections from 6-week-old Pml^+/− and Pml^−/− virgin mammary glands highlighting the increase in the number of ERα⁺ cells. (D) Counts of ERα⁺ cells in Pml^+/− and Pml^−/− virgin mammary glands (n = 3, ***P < 0.01). (E) Gata-3 and Notch-1 mRNA expression analyzed by RT-PCR from the sorted Sca1- cell populations of Pml^+/− and Pml^−/− glands. Control lanes are without RNA (labeled W) and RNA from a 15DG Pml^+/− gland (labeled C). The absence of Notch-1 expression suggests that Stat6 activity is regulating Gata-3 expression.

Discussion

In this study, a physiological function for the PML protein in the development of the mammary gland is presented. Throughout the course of mammary gland development, there exist phases of significant cellular growth, differentiation, and death. Here it is shown that the function of PML in this tissue is to regulate the balance of progenitor cell populations that contribute to the growth and expansion of the mammary epithelium. Previous studies of PML have focused on its role in stress and innate immune responses as well as its role in tumor suppression (1, 3, 18). However, a recent report of a physiological role for PML in regulating hematopoietic stem cell maintenance (8) suggests that further developmental functions may exist. In this study, PML has been shown to affect mammary epithelial differentiation by regulating progenitor cell populations, with the loss of PML resulting in deregulated ductal and alveolar development.

Mammary gland development is regulated by cytokine and hormonal signals, and different members of the Stat family are required throughout this process, as shown by genetic ablation studies in vivo (21–23). The initial observation that PML expression is highly regulated during MGD was followed by the discovery that Stat3 and Stat6 signaling negatively regulates PML expression in this tissue, and this is in keeping with previous evidence that PML is a direct Stat1/2 target (20, 31). PML expression was found to be highest in the virgin mammary gland and at its lowest during lactation. It can be postulated that reduced levels of PML protects the alveolar cells from dying during a time when they are producing a large amount of milk protein. During involution, PML expression was re-induced, resulting in the expression of small “microbodies” of PML which, over time, developed into larger, classical PML-NDs. This re-induction of PML-NDs coincides with massive tissue reorganization and cell death. It is clear, then, that a combination of cytokine and growth factor signaling through the Stat family of signal transducers tunes the levels of PML expression to regulate normal MGD.

Analysis of Pml null mice revealed a novel function in the regulation of branching morphogenesis indicated by aberrant duct size and branch numbers in virgin Pml^−/− mammary glands. Neither an altered proliferative nor apoptotic rate could be detected in the mammary glands of these mice, suggesting that more complex or transient regulatory mechanisms underlie the ductal phenotypes described. A major finding in this study was the identification of skewed mammary gland progenitor cell populations in Pml null animals. The loss of PML was found to result in atypical differentiation of the mammary epithelium, manifesting in abnormal ductal growth and distribution of ER⁺ cells in virgin glands and in delayed functional development during pregnancy, highlighted by reduced β-casein expression as well as lower levels of pStat5 and pStat6. Recent data has highlighted a role for PML in preserving quiescent stem cell niches (8). In this scenario, high levels of PML were used to maintain quiescence. Here, altered distributions of progenitor cells, which may be exquisitely sensitive to stress and cell death, are described. Our demonstration of increased progenitor frequencies in PML-null cells is consistent with stem cell data (8) in which exhaustive cell cycling initially generates elevated levels of stem cells that are depleted at later time-points.

The physiological roles of PML protein are wide ranging, encompassing a multitude of cellular signaling pathways and heterogeneous protein complexes coordinated by PML. Future work to deconstruct the signaling downstream of PML that allows it to control epithelial differentiation will add to our increasing knowledge of the complex regulatory processes that govern ductal development, and will improve our understanding of the precise physiological role of the PML protein.

Materials and Methods

Mice.

Pml^−/− animals backcrossed to the sv129 S2 strain were a kind gift of Paolo Salomoni. Stat6^−/− (23) and BLG-Cre/Stat3^flox/flox conditional knockout (32) mice have previously been described. All animals were maintained in a Biological Services facility at the University of Cambridge and were bred and subjected to listed procedures under the U.K. Home Office guidelines.

Mammary Gland Preparations.

For whole-mount and H&E analysis, glands were prepared as previously described (23).

RNA Preparation and PCR Analysis.

RNA was extracted and real-time detection of cDNA was performed as previously described (23). Primer details are supplied in the SI Text.

Cell Culture.

EpH4 cells were maintained in 1:1 DMEM:F12 medium supplemented with 10% fetal calf serum (FCS, vol/vol). KIM-2 cells (26) were grown to confluency in 1:1 Dulbecco's modified Eagle's medium (DMEM):F12 (Invitrogen) containing 10% FCS (Sigma), 0.8 mM insulin (Sigma), 0.8 mM epidermal growth factor (EGF; Sigma), and 17 mM linoleic acid (Sigma). For differentiation studies, cells were grown to confluency before addition of differentiation media, comprising 1:1 DMEM:F12, 10% FCS, 0.8 mM insulin, 0.2 mM prolactin (Sigma), 1 mM dexamethasone (Sigma), and 17 mM linoleic acid. For activation of Stat signaling, prolactin was used at 5 μg/ml, IL-13 at 40 ng/ml and mOSM at 25 ng/l (R&D Systems).

Immunodetection.

For immunohistochemistry, mammary gland sections were deparaffinized and boiled in 10 mM Tri-sodium citrate buffer, pH 6.0, for 10 minutes. Sections were blocked in 10% normal goat serum (Dako) for 1 hour at room temperature, incubated with primary antibody overnight at 4 °C, and subjected to detection using AlexaFluor-conjugated secondary antibodies (Invitrogen). For immunocytochemistry, cultured cells were fixed in 4% paraformaldehyde, permeablized with 0.25% TritonX-100, blocked with 5% milk in phosphate-buffered saline solution for 1 hour, and immunostained as described above. Immunoblotting was conducted as previously described (23). Antibodies against pStat3, Stat3, pStat5, Stat-5 (Cell Signaling) pStat6, β-actin (Abcam), PML (Alexis), E-cadherin (BD Bioscience), Stat-6, ERα (Sigma), p63, SMA (Neomarkers) CK18 (PROGEN Biotechnik), and transferrin receptor (Zymed) were used to probe for specific antigens.

Flow Cytometry and Colony Assays.

Mammary glands from 8–14-week-old virgin mice were dissected, and mammary epithelial cell suspensions were prepared as previously described (29). Primary antibodies used were as follows: biotinylated anti-CD45, anti-Ter119 and anti-CD31 antibodies; anti-CD24-R-phycoerythrin (PE, clone M1/69, eBioscience), anti-CD49f-AlexaFluor-647 (Clone GoH3, eBioscience), anti-CD49b- Alexa Fluor 488 (Clone HMa2, eBioscience), and Sca1-AF647 (Clone D7, eBioscience). The secondary antibody used was Strepavidin-PE-Texas Red (Invitrogen). Dead cells were excluded by elimination of propidium iodide (PI)-positive cells. For colony-formation assays, freshly sorted cells were plated on irradiated NIH 3T3 feeders (at 10⁴ cells/cm²) in Human NeuroCult NS-A Proliferation Kit (StemCell Technologies Inc. ) supplemented with 10 ng/ml basic fibroblast growth factor (Peprotech), 10 ng/ml EGF, 1:100 dilution of N2 supplement (Invitrogen) and 5% FCS and incubated for another 7 days. Colonies were fixed using ice-cold acetone/methanol (1:1) and visualized using Giemsa stain (Merck).

Acknowledgments

This work was supported by the Breast Cancer Campaign (W.L. and J.S.), an Italian Cancer Research Association grant (to V.P.), Biotechnology and Biological Sciences Research Council (BBSRC) project grants (to T.R., B.J.F., and W.T.K.), the Research Councils UK (T.R.), a BBSRC Research Development Fellowship (to C.J.W.), and the Medical Research Council (P.S.).

Footnotes

⁴To whom correspondence should be addressed. E-mail: cjw53{at}mole.bio.cam.ac.uk
Author contributions: C.J.W. designed research; W.L., B.J.F., W.T.K., M.T., J.S., and C.J.W. performed research; B.J.F., W.T.K., J.S., V.P., T.R., and P.S. contributed new reagents/analytic tools; W.L., B.J.F., W.T.K., J.S., T.R., and C.J.W. analyzed data; and W.L., B.J.F., W.T.K., T.R., and C.J.W. wrote the paper.
↵²Present address: Department of Virology, Imperial College, Norfolk Place, London W2 1PG, United Kingdom.
↵³Present address: Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, United Kingdom.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. M.B. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/cgi/content/full/0807640106/DCSupplemental.

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(2007) PML and PML nuclear bodies: Implications in antiviral defense. Biochimie 89:819–830.
OpenUrl CrossRef PubMed
↵
1. Levy DE,
2. Darnell JE, Jr
(2002) Stats: Transcriptional control and biological impact. Nature Rev Mol Cell Biol 3:651–662.
OpenUrl CrossRef PubMed
↵
1. Choi YH,
2. Bernardi R,
3. Pandolfi PP,
4. Benveniste EN
(2006) The promyelocytic leukemia protein functions as a negative regulator of IFN-gamma signaling. Proc Natl Acad Sci 103:18715–18720.
OpenUrl Abstract/FREE Full Text
↵
1. Watson CJ,
2. Neoh K
(2008) The Stat family of transcription factors have diverse roles in mammary gland development. Semin Cell Dev Biol 19:401–406.
OpenUrl CrossRef PubMed
↵
1. Hennighausen L,
2. Robinson GW,
3. Wagner KU,
4. Liu X
(1997) Developing a mammary gland is a Stat affair. J Mammary Gland Biol Neoplasia 2:365–372.
OpenUrl CrossRef PubMed
↵
1. Khaled WT,
2. et al.
(2007) The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development. Development 134:2739–2750.
OpenUrl Abstract/FREE Full Text
↵
1. Liu X,
2. et al.
(1997) Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186.
OpenUrl Abstract/FREE Full Text
↵
1. Kritikou EA,
2. et al.
(2003) A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland. Development 130:3459–3468.
OpenUrl Abstract/FREE Full Text
↵
1. Gordon KE,
2. et al.
(2000) A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland. Breast Cancer Res 2:222–235.
OpenUrl CrossRef PubMed
↵
1. Chapman RS,
2. et al.
(1999) Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev 13:2604–2616.
OpenUrl Abstract/FREE Full Text
↵
1. Raouf A,
2. et al.
(2008) Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell 3:109–118.
OpenUrl CrossRef PubMed
↵
1. Stingl J,
2. et al.
(2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997.
OpenUrl PubMed
↵
1. Kouros-Mehr H,
2. Slorach EM,
3. Sternlicht MD,
4. Werb Z
(2006) GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell 127:1041–1055.
OpenUrl CrossRef PubMed
↵
1. Vlasakova J,
2. et al.
(2007) Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109:1373–1380.
OpenUrl Abstract/FREE Full Text
↵
1. Alonzi T,
2. et al.
(2001) Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver. Mol Cell Biol 21:1621–1632.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 106 (12)

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[1] ↵
Salomoni P,
Ferguson BJ,
Wyllie AH,
Rich T
(2008) New insights into the role of PML in tumour suppression. Cell Res 18:622–640.
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[5] Rich T

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[56] et al.

[57] ↵
Everett RD,
Chelbi-Alix MK
(2007) PML and PML nuclear bodies: Implications in antiviral defense. Biochimie 89:819–830.
OpenUrl CrossRef PubMed

[58] Everett RD,

[59] Chelbi-Alix MK

[60] ↵
Levy DE,
Darnell JE, Jr
(2002) Stats: Transcriptional control and biological impact. Nature Rev Mol Cell Biol 3:651–662.
OpenUrl CrossRef PubMed

[61] Levy DE,

[62] Darnell JE, Jr

[63] ↵
Choi YH,
Bernardi R,
Pandolfi PP,
Benveniste EN
(2006) The promyelocytic leukemia protein functions as a negative regulator of IFN-gamma signaling. Proc Natl Acad Sci 103:18715–18720.
OpenUrl Abstract/FREE Full Text

[64] Choi YH,

[65] Bernardi R,

[66] Pandolfi PP,

[67] Benveniste EN

[68] ↵
Watson CJ,
Neoh K
(2008) The Stat family of transcription factors have diverse roles in mammary gland development. Semin Cell Dev Biol 19:401–406.
OpenUrl CrossRef PubMed

[69] Watson CJ,

[70] Neoh K

[71] ↵
Hennighausen L,
Robinson GW,
Wagner KU,
Liu X
(1997) Developing a mammary gland is a Stat affair. J Mammary Gland Biol Neoplasia 2:365–372.
OpenUrl CrossRef PubMed

[72] Hennighausen L,

[73] Robinson GW,

[74] Wagner KU,

[75] Liu X

[76] ↵
Khaled WT,
et al.
(2007) The IL-4/IL-13/Stat6 signalling pathway promotes luminal mammary epithelial cell development. Development 134:2739–2750.
OpenUrl Abstract/FREE Full Text

[77] Khaled WT,

[78] et al.

[79] ↵
Liu X,
et al.
(1997) Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186.
OpenUrl Abstract/FREE Full Text

[80] Liu X,

[81] et al.

[82] ↵
Kritikou EA,
et al.
(2003) A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland. Development 130:3459–3468.
OpenUrl Abstract/FREE Full Text

[83] Kritikou EA,

[84] et al.

[85] ↵
Gordon KE,
et al.
(2000) A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland. Breast Cancer Res 2:222–235.
OpenUrl CrossRef PubMed

[86] Gordon KE,

[87] et al.

[88] ↵
Chapman RS,
et al.
(1999) Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev 13:2604–2616.
OpenUrl Abstract/FREE Full Text

[89] Chapman RS,

[90] et al.

[91] ↵
Raouf A,
et al.
(2008) Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell 3:109–118.
OpenUrl CrossRef PubMed

[92] Raouf A,

[93] et al.

[94] ↵
Stingl J,
et al.
(2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997.
OpenUrl PubMed

[95] Stingl J,

[96] et al.

[97] ↵
Kouros-Mehr H,
Slorach EM,
Sternlicht MD,
Werb Z
(2006) GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell 127:1041–1055.
OpenUrl CrossRef PubMed

[98] Kouros-Mehr H,

[99] Slorach EM,

[100] Sternlicht MD,

[101] Werb Z

[102] ↵
Vlasakova J,
et al.
(2007) Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109:1373–1380.
OpenUrl Abstract/FREE Full Text

[103] Vlasakova J,

[104] et al.

[105] ↵
Alonzi T,
et al.
(2001) Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver. Mol Cell Biol 21:1621–1632.
OpenUrl Abstract/FREE Full Text

[106] Alonzi T,

[107] et al.

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