Expression of Wnt5a Is Downregulated by Extracellular Matrix and Mutated c-Ha-ras in the Human Mammary Epithelial Cell Line MCF-10A

Wnt genes are involved in tumour growth and regulate cell adhesion. Some (Wnt5a and Wnt7b) are more highly expressed in human breast cancer compared to normal tissues. Wnt5a is involved in the regulation of cell movement inXenopusand is upregulated in several human cancers. Factors regulating Wnt gene expression in human breast epithelium are poorly understood, but c-erbB2 is amplified in many breast cancers and associated with rapid growth and metastasis, as is high expression of c-Ha-ras. To further understand the regulation of Wnt gene expression, this study investigated the effect of proto-oncogenes c-Ha-ras and c-erbB2, and collagen on Wnt mRNA expression, in a normal spontaneously immortalised human mammary epithelial cell line MCF-10A. Out of nine human Wnt genes investigated, Wnt5a and Wnt7b were expressed in the parental cell line, and neomycin-, c-Ha-ras- and c-erbB2-transfected cell lines. The level of Wnt5a mRNA expression was decreased 40-fold and 3-fold when parental cells were grown on collagen and in collagen, respectively. This downregulation correlated with cell branching. However, Wnt7b was not regulated by collagen. In the presence of activated c-Ha-ras, the level of Wnt5a mRNA expression was markedly decreased (> 200-fold) and cell growth rate was elevated. When treated with p21^rasinhibitor, BZA-5B, there was a moderate reversal of Wnt5a mRNA expression (2-fold) with a parallel decrease in cell growth. The data indicate that c-Ha-ras is an upstream inhibitory regulator of Wnt5a, and provide further evidence of an inverse relationship between Wnt5a mRNA expression and cell branching. This demonstrates selectivity of regulation of individual members of the Wnt gene family by the ras pathway. Overexpression of c-erbB2 had no effect on Wnt5a or Wnt7b mRNA expression. Thus, extracellular matrix and ras regulate Wnt5a, providing a mechanism for feedback of morphogenetic movements, which is relevant also to cancer biology.     

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.     

Stable Polarized Expression of hCAT-1 in an Epithelial Cell Line

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.     

renalase在人近曲肾小管上皮细胞系的表达与分泌

目的:探讨renalase在人近曲肾小管上皮细胞系(HK-2)的表达与分泌,为进一步研究细胞水平renalase及其通路建立稳定的实验平台。方法:以HK-2细胞系作为研究材料。①应用Westernblot方法检测renalase蛋白的表达。②用real-timePCR方法检测renalasemRNA表达的变化。③用ELISA方法检测细胞上清液中renalase的浓度。结果:在mRNA水平及蛋白水平均检测到renalase表达。结论:首次在mRNA水平及蛋白水平证实了HK-2细胞能够表达renalase,为进一步研究儿茶酚胺或缺血缺氧刺激下细胞renalase的表达奠定了基础。     

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.     

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.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

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.     

Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.     

Expression and Localization of GEN1 in Mouse Mammary Epithelial Cells

GEN1, a Holliday junction resolvase, is involved in homologous repair of DNA double strand break and in maintaining centrosome integrity. Although GEN1 mutants have been reported in breast cancer patients and cell lines, little is currently known about the functions of GEN1 in the development and oncogenic transformation of mammary gland. In the present study, we demonstrate that GEN1 expression is correlated with mammary epithelial cell proliferation, differentiation in various physiological stages as well as casein. By immunofluorescence analysis, the centrosomal association of GEN1 is confirmed in mammary epithelial cells. Additionally, GEN1 is likely involved in DNA damage response of breast cancer cell lines. These results suggest that GEN1 may play an important role in the development of mammary gland; its response upon DNA damage indicates that GEN1 gene alteration may contribute to breast cancer formation.     

Establishment of Mammary Gland Model In Vitro: Culture and Evaluation of a Yak Mammary Epithelial Cell Line

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.     

Hyposmotically-Activated Efflux of L-Carnitine from a Human Mammary Cancer Cell Line

Cell-swelling, induced by a hyposmotic challenge, stimulated the efflux of L-carnitine from a human mammary cancer cell line, MDA-MB-231. The response was dependent upon the extent of the osmotic shock. Hyposmotically-activated L-carnitine efflux was inhibited by the anion transport blocker diiodosalicylate. The efflux of taurine from MDA-MB-231 cells was also stimulated by a hyposmotic shock via a pathway sensitive to diiodosalicylate. L-carnitine efflux from MDA-MB-231 cells was stimulated by isosmotic swelling in a manner which was inhibited by diiodosalicylate. The results suggest that L-carnitine may exit cells via a volume-sensitive pathway: it is possible that L-carnitine efflux may utilize the same pathway as amino acids. The efflux of L-carnitine via this route could have a major effect on the intracellular concentration of L-carnitine and could facilitate transepithelial L-carnitine transport.     

Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma

Background

Gliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function.

Methods and Results

To better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs.

Conclusions

Restoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs.     

Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.     

JAK3蛋白在鼻咽癌细胞中参与STAT活化并受EB病毒潜伏膜蛋白1调控

为了探讨在鼻咽癌细胞（NPC）中是否存在EB病毒潜伏膜蛋白1（LMP1）/JAK3/STAT信号传导途径,首先采用RT-PCR方法对NPC JAK家族4种成员检测,发现在两株鼻咽癌细胞株CNE1和HNE2中该家族4种成员均有表达.选择最有可能与LMP1相互作用的JAK家族成员JAK3作为我们的研究对象.利用已建立的一株受四环素衍生物强力霉素(doxycycline,Dox)调控的LMP1表达的鼻咽癌细胞系(Tet-on-LMP1 HNE2),诱导Tet-on-LMP1 HNE2细胞LMP1动态表达,蛋白质印迹发现JAK3的表达方式呈剂量和时间依赖性.采用瞬间转染方法将STAT报告基因质粒（GRR-Luc）转染入pTet-on-LMP1 HNE2细胞中,不同剂量的Dox促使LMP1的表达可以激活STAT报告基因活性,在0.06mg/L Dox诱导36 h时,STAT活性最高.在该条件下,加入3 μmol/L JAK3特异性抑制剂WHI-P131时,可抑制STAT的活性.结果表明：JAK3的表达在NPC细胞中受LMP1的调控,LMP1可以通过调控JAK3的表达参与STAT的活化.在鼻咽癌细胞中存在LMP1/JAK3/STAT信号途径并可能在其发生发展过程中起重要作用.     

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1^TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1^TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1^TG bone marrow cells into non-transgenic (AEBP1^NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1^TG mammary macrophages and epithelium. Shh expression was induced in AEBP1^TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1^TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1^TG peritoneal macrophages. The conditioned AEBP1^TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1^TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.