WDNM1 is Associated with Differentiation and Apoptosis of Mammary Epithelial Cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFα treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFα induced WDNM1 expression showed the NFκB-dependent mechanism since it's expression was abrogated in IκBαM (super-repressor of NFκB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFα-NFκB signal pathway, is associated with HC11 cell apoptosis.     

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres ¹. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo ². The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.Download video file.^{(57M, mp4)}     

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.     

The Impact of ABCG2 on Bovine Mammary Epithelial Cell Proliferation

The ATP-binding cassette transporter, ABCG2, has been identified as a gene of significance in the regulation of bovine lactation by a number of gene mapping studies yet its role in lactational physiology remains unclear. We have used the potent ABCG2 specific inhibitor, Ko143, to investigate role of ABCG2 in primary bovine mammary epithelial cell (BMEC) proliferation and differentiation. After incubation with Ko143, the proliferation rate of BMECs was reduced at 48 and 72 hours by up to 80% (P?相似文献   

The Impact of ABCG2 on Bovine Mammary Epithelial Cell Proliferation

The ATP-binding cassette transporter, ABCG2, has been identified as a gene of significance in the regulation of bovine lactation by a number of gene mapping studies yet its role in lactational physiology remains unclear. We have used the potent ABCG2 specific inhibitor, Ko143, to investigate role of ABCG2 in primary bovine mammary epithelial cell (BMEC) proliferation and differentiation. After incubation with Ko143, the proliferation rate of BMECs was reduced at 48 and 72 hours by up to 80% (P < 0.001), and the effect was dose-dependent (approximately 40% with 10 nM Ko143 and 80% with 20 nM Ko143). Morphological changes in BMEC mammosphere formation were not observed when co-incubated with Ko143. Our results suggested that ABCG2 plays a role in mammary epithelial cell proliferation and that functional polymorphisms in this gene may influence the cellular compartment of the mammary gland and potentially milk production.     

Murine Mammary Epithelial Stem Cells: Discovery,Function, and Current Status

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.Before the 1980s there was little if any thought that the epithelium in murine mammary glands might be engendered by or supported by a mammary epithelial specific stem cell. In 1980, Rudland et al. wrote a review entitled “Stem cells in rat mammary development and cancer: A review” and noted that dimethylbenz [α] anthracene (DMBA)-induced rat carcinomas contained all three main types of epithelium found in the normal rat gland, those lining the ductal lumina, those lining the alveolar lumina, and myoepithelial cells (Rudland et al. 1980). They suggested, based on the two types of morphologically distinct epithelial (luminal and myoepithelial) cancer cells in the clonally derived Rama 25 cell line, that a single cell might give rise to both types and this also held true when these cells were inoculated into hosts and produced tumors. Williams and Daniel (Williams and Daniel 1983) suggested that the cap cells at the tip of the growing ducts in the mouse could give rise to both luminal and myoepithelial cells during ductal morphogenesis. However, no direct evidence that a single cell could produce both epithelial cell types in vivo was available. Nevertheless, in retrospect there was evidence that full regenerative activity for mammary epithelial existed in every part of the adult mammary epithelial tree.The experiments that originally showed the potential existence of stem cells in the mouse mammary gland were the pioneering studies of DeOme and his students, Les Faulkin and Charles Daniel. The approach they developed and optimized was serial transplantation of normal mammary gland into the cleared mammary fat pad of syngeneic mice (Deome et al. 1959; Faulkin and Deome 1960). The cleared mammary fat pad allowed the transplantation and growth of normal mammary cells into their normal anatomical site and under the influence of a normal physiological environment. Using this method, DeOme and coworkers showed that all portions of the normal mammary gland contains cells that will grow and fill the fat pad with a normal ductal mammary tree and respond to hormones with a normal differentiation program (Daniel 1975; Daniel et al. 1975). The progeny of the transplanted cells could be serially transplanted into the appropriate recipients for multiple times; however, unlike preneoplastic or neoplastic cells, the normal cells always senesced after multiple serial transplants, generally five to eight transplant generations (Daniel 1975). This was interpreted as indicating mammary stem cells possessed a finite proliferative activity (i.e., life span). This finite life span was a fundamental difference between normal and preneoplastic/neoplastic mammary cells. Cells with an indefinite in vivo life span (i.e., immortalized) have been identified in numerous mammary model systems, including MMTV-induced alveolar hyperplasia''s (Callahan and Smith 2000), chemical carcinogen-induced ductal and alveolar hyperplasia''s (Smith et al. 1978, 1980), hormonally induced alveolar hyperplasia, spontaneously immortalized ductal hyperplasia''s (Medina 2000, 2002), and cells containing specific genetic alterations (i.e., p53 deletion, Polyoma mT antigen) (Maglione et al. 2001; Medina et al. 2002).Subsequent studies showed that stem cells were located along the entire mammary tree and represented in all the different developmental states of the mammary gland. These stages included primary and tertiary ducts from 6- and 16-wk virgin glands, uniparous and multiparous regressed gland, 15-d pregnant and 10-d lactating glands (Smith and Medina 1988). Host age and reproductive history had little influence on the frequency of stem cells as measured by percent successful takes and life span assay (Young et al. 1971; Smith and Medina 1988). Mammary cells taken from 26-mo-old virgin mice had the same transplant potential as cells taken from 3-wk-old mice. Cell populations, from both, senesced after five transplant generations. Similarly, mammary cells in 12-mo-old multiparous mice had the same serial transplant potential as cells from 3-wk-old virgin mice (Young et al. 1971). Finally, continuous hormone stimulation did not induce additional loss of ductal growth potential. These results have important implications for understanding the role of mammary stem cells in normal mammary development because they emphasize that the mammary stem cell is a relatively quiescent cell that is only activated under conditions of gland repopulation (i.e., fetal growth stage and pubertal growth phase).     

Induction of Biochemical Differentiation in Three-Dimensional Collagen Cultures of Mammary Epithelial Cells from Virgin Mice

In order to reduce cellular complexity in the study of the controls of the biochemical differentiation of mammary gland epithelium, approximately 100-fold purified epithelial cells from the mammary glands of virgin BALB/c mice were grown in three-dimensional collagen gels, and formed colonies that resembled mammary ductules. Here we report the induction of a biochemical differentiation in these purified epithelial cells in response to appropriate hormonal signals, starting from the state in the virgin mammary gland and ending with the stage characteristic of lactation. Induction of the synthesis of caseins was examined as a marker of mammary functional differentiation using sensitive immunologic autoradiography. The cells were maximally induced by the combination of the hormones, insulin, prolactin, aldosterone, and hydrocortisone, in both serum-containing and essentially serum-free media. The induction required insulin and prolactin, and was enhanced by the presence of the steroids. The cellular distribution of the induction was general, inasmuch as three-quarters of the hormone-stimulated cells were casein-positive according to immunocytochemistry. In order to assess the role of the three-dimensional conformation in the induction process, the purified mammary epithelial cells were grown as monolayers on plastic and collagen-coated surfaces. In these two-dimensional cultures, the synthesis of casein was not induced, suggesting that cell shape, orientation, and multicellular organization are important parameters in the hormonal induction of the biochemical differentiation. The finding of the induction of differentiation-specific proteins in cultures of purified epithelial cells from virgin glands allows examination of the molecular mechanisms involved in the complete induction process in the virtual absence of fat cells, fibroblasts, and the complex assortment of biochemical constituents of the mammary fat pad.     

Regulation of Autophagy in Bovine Mammary Epithelial Cells

The bovine mammary gland undergoes intensive remodeling during the lactation cycle, and the escalation of this process is observed during dry periods. The main type of cell death responsible for bovine mammary gland involution is apoptosis; however, there are also a lot of cells exhibiting morphological features of autophagy during drying off. Our in vitro and in vivo studies of bovine mammary gland physiology suggest that the enhanced process of autophagy, observed at the end of lactation and during dry periods, is the result of: (1) decreased level of lactogenic hormones (GH, IGF-I), (2) decreased GH-R and IGF-IRα expression, (3) increased expression of auto/paracrine apoptogenic peptides (IGFBPs, TGF-β1), (4) increased influence of sex steroids (17β-estradiol and progesterone) and (5) enhanced competition between the intensively developing fetus and the mother organism for nutritional and bioactive compounds. The above conditions may create a state of temporary malnutrition of mammary epithelial cells, which forces the cells to the induction of autophagy, as a mechanism for stabilizing intracellular supplies of energy and amino acids, especially during the enhanced activity of apoptogenic factors.

Addendum to:

Apoptosis and Autophagy in Mammary Gland Remodeling and Breast Cancer Chemotherapy

T. Motyl, B. Gajkowska, J. Zarzyńska, M. Gajewska and M. Lamparska-Przybysz

J Physiol Pharmacol 2006; 57:17-32     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

Subcellular Localization of p44/WDR77 Determines Proliferation and Differentiation of Prostate Epithelial Cells

The molecular mechanism that controls the proliferation and differentiation of prostate epithelial cells is currently unknown. We previously identified a 44-kDa protein (p44/wdr77) as an androgen receptor-interacting protein that regulates a set of androgen receptor target genes in prostate epithelial cells and prostate cancer. In this study, we found that p44 localizes in the cytoplasm of prostate epithelial cells at the early stage of prostate development when cells are proliferating, and its nuclear translocation is associated with cellular and functional differentiation in adult prostate tissue. We further demonstrated that cytoplasmic p44 protein is essential for proliferation of prostate epithelial cells, whereas nuclear p44 is required for cell differentiation and prostate- specific protein secretion. These studies suggest a novel mechanism by which proliferation and differentiation of prostate epithelial cells are controlled by p44’s location in the cell.     

Brca1-Deficient Murine Mammary Epithelial Cells have Increased Sensitivity to CDDP and MMS

In this report we describe the isolation of an isogenic pair of Brca1+/+ and Brca1-/-murine mammary epithelial cells (MMECs). These cells were isolated from Brca1conditional knock out mice which contained loxP sites flanking exon 11 of the Brca1gene (Brca1fl/fl) and then immortalized by infection with HPV-16E6 retrovirus to degradep53 protein. Brca1-/- MMECs were generated by deletion of exon 11 followingtransduction of Brca1fl/fl MMECs with a retroviral vector expressing Cre recombinase.Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride(CDDP) and methylmethane sulfonate (MMS). The Brca1+/+ and Brca1-/- MMECS is theonly known pair of isogenic mammary epithelial cell lines. The understanding of themechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cellswould be very important in understanding how BRCA1-deficiency plays out in tissuespecific breast cancer chemotherapy. These studies support the role of BRCA1 in theCDDP-induced and MMS-induced DNA damage and repair by p53-independentpathways.     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

Differential Sensitivity of Epithelial Cells to Extracellular Matrix in Polarity Establishment

Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.     

EGF Modulates Expression of STAT5 in Mammary Epithelial Cells

HC11 mouse mammary epithelial cells are capable of differentiatingin vitro.By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiationin vivo.This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seenin vivo.This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.     

小鼠和兔原代乳腺上皮细胞的培养

目的：对小鼠和兔的原代乳腺上皮细胞进行培养。方法：取小鼠和免的乳腺组织,用加葡萄糖的PBS稀释胶原酶Ⅲ作消化液、分三个阶段进行消化,选择离心后的小细胞团,用含激素的培养液培养。结果：在基本培养液中添加10ng／ml的表皮生长因子和5μg／ml的胰岛素可以使乳腺上皮细胞富集．结论：成功地培养出了小鼠和免的原代乳腺上皮细胞。     

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.     

Degree of Differentiation in Nonproliferating Cells of Mammary Carcinoma

A pool of nondividing tumour cells, believed to be nonproliferating stem cells (G^o), has been identified in mammary adenocarcinomas of mice. To determine whether or not some of these cells might be postmitotic differentiated progeny of stem cells, mice bearing spontaneous tumours were either pulsed or perfused with tritium-labelled thymidine for five days, and light and electron autoradiography was performed using standard methods. Unlabelled cells (that had not synthesized DNA for five days) had a range of differentiation, but many of the cells were well differentiated. This observation supports the concept that many of the non-proliferating tumour cells were postmitotic as a result of differentiation and the balance were nonproliferating stem cells (G^o). The latter presumably did not divide because of environmental conditions.