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Sample records for bovine mammary epithelial

  1. Characterization of an epithelial cell line from bovine mammary gland.

    Science.gov (United States)

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  2. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica;

    2008-01-01

    A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermicline and beta-phenylethylamine on mammary epithelial cells. Each biogenic amine was diluted in several concentratio...

  3. Triennial Lactation Symposium: Bovine mammary epithelial cell lineages and parenchymal development.

    Science.gov (United States)

    Ellis, S; Akers, R M; Capuco, A V; Safayi, S

    2012-05-01

    Mammary development proceeds from an aggregation of cells in the ventral ectoderm to the establishment of an elaborate tree of alveoli, ducts, and cisternae. However, despite abundant data on endocrine regulation of ruminant mammary growth, we know comparatively little about cell lineages, expression of differentiation markers, and plasticity in mammary cell phenotype. Histologic analyses have revealed cell populations with distinct histochemical profiles, but functional assessment of cell populations during development has been limited to analysis of proliferation and frequency estimations of morphotypes. The lack of transplantation models, limited availability of validated antibodies with reactivity to bovine antigens, and similar technical challenges have generally hindered the pace of discovery, but the application of new technologies such as laser microdissection, transcriptional profiling, and multispectral image analysis are yielding important clues into bovine mammary cell ontogeny and developmental regulation. Our analyses have shown that prepubertal ovariectomy affects epithelial architecture, increases the proportion of cells expressing the estrogen receptor, and increases myoepithelial cell development, all concomitant with a dramatic reduction in the mass of parenchymal tissue. Our observations point to a dual role for ovarian secretions in the control of not only the rate of epithelial development, but also the nature of the parenchymal development. The balance of stimulus and inhibition pathways cooperatively regulates mammary growth. The increased reliance on objective staining analyses and quantitative approaches will ensure broader repeatability, application, and extension of the findings regarding the impact of the ovary and other regulatory entities and factors. Advances in understanding the ontogeny of mammary epithelial cells, coupled with established and increasing knowledge of endocrine factors affecting mammary development, may yield

  4. Protection of Bovine Mammary Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Cell Damage by Resveratrol

    OpenAIRE

    Xiaolu Jin; Kai Wang; Hongyun Liu; Fuliang Hu; Fengqi Zhao; Jianxin Liu

    2016-01-01

    The mammary epithelial cells (MECs) of high-producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide- (H2O2-) induced OS in cultured bovine MECs (MAC-T). Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after ...

  5. Bovine Mammary Epithelial Cell Lineages and Parenchymal Development

    Science.gov (United States)

    Mammary development proceeds from an aggregation of cells in the ventral ectoderm to the establishment of an elaborate tree of alveoli, ducts, and cisternae. However, despite abundant data on endocrine regulation of ruminant mammary growth, we know comparatively little about cell lineages, express...

  6. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh

    2013-10-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages.

    Science.gov (United States)

    Günther, Juliane; Czabanska, Anna; Bauer, Isabel; Leigh, James A; Holst, Otto; Seyfert, Hans-Martin

    2016-01-01

    Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis. PMID:26738804

  8. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    Directory of Open Access Journals (Sweden)

    Babak Qasemi-Panahi

    2013-02-01

    Full Text Available Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1 on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 μL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  9. Mammary epithelial cell

    DEFF Research Database (Denmark)

    Kass, Laura; Erler, Janine Terra; Dembo, Micah;

    2007-01-01

    mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal...... organization, and promote cell invasion and survival. In this review, we discuss the role of stromal-epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit...

  10. Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid.

    Science.gov (United States)

    Wellnitz, Olga; Zbinden, Christina; Huang, Xiao; Bruckmaier, Rupert M

    2016-06-01

    In the mammary gland, the blood-milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood-milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood-milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS. PMID:27060811

  11. Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Maria Hillreiner

    Full Text Available Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC challenged with the mastitis pathogen Escherichia coli (E. coli. Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.

  12. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

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    Li Qianqian

    2010-10-01

    Full Text Available Abstract Background Spontaneous immortalisation of cultured mammary epithelial cells (MECs is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs and the changes in gene expression associated with BME65Cs cells. Results BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous bTERT (bovine Telomerase Reverse Transcriptase and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line. In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16INK4a was downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene c-Myc, along with an undetectable level of breast tumor-related gene Bag-1 and TRPS-1, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, DNMT1 is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of p53 and p16INK4a in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of p53 or p16INK4a. Conclusions Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and

  13. 奶牛乳腺上皮细胞系的培养与鉴定%Culture and Identification of the Bovine Mammary Epithelial Cell Line

    Institute of Scientific and Technical Information of China (English)

    詹康; 贡笑笑; 左晓昕; 陈银银; 占今舜; 赵国琦

    2015-01-01

    本试验旨在培养功能性奶牛乳腺上皮细胞系,为奶牛泌乳调控和奶牛乳房炎发病机制研究提供功能性的细胞模型。采用组织块细胞培养法来分离纯化并鉴定奶牛乳腺上皮细胞;采用有限稀释法单克隆奶牛乳腺上皮细胞;采用噻唑蓝( MTT)法来分析奶牛乳腺上皮细胞的生长曲线是否为正常的“S”形;观察细胞角蛋白18免疫荧光来证明所培养的细胞为上皮型;选择培养至10和20代的奶牛乳腺上皮细胞进行染色体核型分析。结果表明:1)利用组织块细胞分离法能够成功获得奶牛乳腺上皮细胞并传至20代。2)培养5~6 d成纤维细胞迅速增殖且周围分裂出少量的上皮细胞。培养8d奶牛乳腺上皮细胞迅速增殖,形成岛屿状集落,呈单层“鹅卵石”和“铺路石”形态生长。3)奶牛乳腺上皮细胞角蛋白18鉴定为阳性。4)培养至10和20代的奶牛乳腺上皮细胞染色体数为60条,具有正常的细胞二倍体核型。综上所述,采用组织块培养细胞能够获得具有稳定性、功能性的奶牛乳腺上皮细胞,但不是永生化细胞。%To establish a functional model of bovine mammary epithelial cell line for the study of lactation reg-ulation, pathogenesis of mastitis, this research applied tissue culture to isolate and culture bovine mammary epi-thelial cells; the limiting dilution method to purify bovine mammary epithelial cells; methyl thiazolyl tetrazoli-um ( MTT) method was used to identify whether the growth curve of bovine mammary epithelial cells was‘S’ type or not;cytokeratin 18 immunofluorescence was observed to prove that the cells were epithelial type;kary-otype analysis was carried out on bovine mammary epithelial cells in passages 10 and 20. The results showed as follows:1) bovine mammary epithelial cells could be successfully cultured and passaged 20 generations by tis-sue culture method. 2) At 5 to 6 days of cultivation, the

  14. IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

    Science.gov (United States)

    Gajewska, Małgorzata; Motyl, Tomasz

    2004-10-01

    TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway. PMID:15556067

  15. Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells.

    Science.gov (United States)

    Li, Xiaojuan; Li, Lian; Sun, Yu; Wu, Jie; Wang, Genlin

    2016-05-01

    Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala → Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 μg/mL) for 12 h, RBLBP of 5 μg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 μg/mL of mutant LBP and the BMECs only treated with 10 μg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 μg/mL of wild LBP and 10 μg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands. PMID:26813383

  16. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

    Science.gov (United States)

    Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

    2016-01-01

    Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis. PMID:27433029

  17. Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior.

    Science.gov (United States)

    Souza, F N; Piepers, S; Della Libera, A M M P; Heinemann, M B; Cerqueira, M M O P; De Vliegher, S

    2016-04-01

    Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifer's teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior. PMID:26830736

  18. Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

    Science.gov (United States)

    Medina-Estrada, Ivan; López-Meza, Joel E.

    2016-01-01

    17β-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5β1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1β (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1β. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization. PMID:27034592

  19. Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

    Science.gov (United States)

    Díaz-Murillo, Violeta; Medina-Estrada, Ivan; López-Meza, Joel E; Ochoa-Zarzosa, Alejandra

    2016-04-01

    β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian

  20. Functional Interactions between 17β-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures

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    Katarzyna Zielniok

    2014-01-01

    Full Text Available Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2 and progesterone (P4. Mammary epithelial cells (MECs cultured on reconstituted basement membrane (rBM form three-dimensional (3D acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR involved in autophagy induction.

  1. Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

    OpenAIRE

    Ontsouka, Edgar; Bertschi, Janique Sabina; Huang, Xiao; Lüthi, Michael; Müller, Stefan Jürg; Albrecht, Christiane

    2016-01-01

    BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alv...

  2. Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

    OpenAIRE

    Ontsouka, Edgar Corneille; Bertschi, Janique Sabina; Huang, Xiao; Lüthi, Michael; Müller, Stefan; Albrecht, Christiane

    2016-01-01

    Background Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein–Friesian (bMECCH) cows, and of primary bovine mammary alveola...

  3. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel); The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem (Israel); Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel)

    2014-10-15

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth.

  4. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    International Nuclear Information System (INIS)

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth

  5. Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells

    Science.gov (United States)

    Xia, X-j; Gao, Y-y; Zhang, J; Wang, L; Zhao, S; Che, Y-y; Ao, C-j; Yang, H-j; Wang, J-q; Lei, L-c

    2016-01-01

    Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-γ has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-γ can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-γ treatment, autophagy and malignant transformation and of the mechanism underlying IFN-γ-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-γ levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-γ. In vitro, autophagy was clearly induced in primary BMECs by IFN-γ within 24 h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-γ promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-γ-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet. PMID:27551491

  6. Bovine mammary stem cells: Transcriptome profiling and the stem cell niche

    Science.gov (United States)

    Identification and transcriptome analysis of mammary stem cells (MaSC) are important steps toward understanding the molecular basis of mammary epithelial growth, homeostasis and tissue repair. Our objective was to evaluate the molecular profiles of four categories of cells within the bovine mammary ...

  7. Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains.

    Science.gov (United States)

    Malvisi, M; Stuknytė, M; Magro, G; Minozzi, G; Giardini, A; De Noni, I; Piccinini, R

    2016-03-01

    Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked

  8. Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

    OpenAIRE

    Gat Rauner; Amos Leviav; Eliezer Mavor; Itamar Barash

    2013-01-01

    Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization...

  9. Effect of the ratios of unsaturated fatty acids on the expressions of genes related to fat and protein in the bovine mammary epithelial cells.

    Science.gov (United States)

    Sheng, R; Yan, S M; Qi, L Z; Zhao, Y L

    2015-04-01

    The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 μM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future

  10. Deep Sequencing and Screening of Differentially Expressed MicroRNAs Related to Milk Fat Metabolism in Bovine Primary Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Binglei Shen

    2016-02-01

    Full Text Available Milk fat is a key factor affecting milk quality and is also a major trait targeted in dairy cow breeding. To determine how the synthesis and the metabolism of lipids in bovine milk is regulated at the miRNA level, primary mammary epithelial cells (pMEC derived from two Chinese Holstein dairy cows that produced extreme differences in milk fat percentage were cultured by the method of tissue nubbles culture. Small RNA libraries were constructed from each of the two pMEC groups, and Solexa sequencing and bioinformatics analysis were then used to determine the abundance of miRNAs and their differential expression pattern between pMECs. Target genes and functional prediction of differentially expressed miRNAs by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis illustrated their roles in milk fat metabolism. Results show that a total of 292 known miRNAs and 116 novel miRNAs were detected in both pMECs. Identification of known and novel miRNA candidates demonstrated the feasibility and sensitivity of sequencing at the cellular level. Additionally, 97 miRNAs were significantly differentially expressed between the pMECs. Finally, three miRNAs including bta-miR-33a, bta-miR-152 and bta-miR-224 whose predicted target genes were annotated to the pathway of lipid metabolism were screened and verified by real-time qPCR and Western-blotting experiments. This study is the first comparative profiling of the miRNA transcriptome in pMECs that produce different milk fat content.

  11. The activation of the TLR2/p38 pathway by sodium butyrate in bovine mammary epithelial cells is involved in the reduction of Staphylococcus aureus internalization.

    Science.gov (United States)

    Alva-Murillo, Nayeli; Medina-Estrada, Ivan; Báez-Magaña, Marisol; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2015-12-01

    Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1β and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen

  12. Deep sequencing-based transcriptional analysis of bovine mammary epithelial cells gene expression in response to in vitro infection with Staphylococcus aureus stains.

    Directory of Open Access Journals (Sweden)

    Xiao Wang

    Full Text Available Staphylococcus aureus (S. aureus is an important etiological organism in chronic and subclinical mastitis in lactating cows. Given the fundamental role the primary bovine mammary epithelial cells (pBMECs play as a major first line of defense against invading pathogens, their interactions with S. aureus was hypothesized to be crucial to the establishment of the latter's infection process. This hypothesis was tested by investigating the global transcriptional responses of pBMECs to three S. aureus strains (S56,S178 and S36 with different virulent factors, using a tag-based high-throughput transcriptome sequencing technique. Approximately 4.9 million total sequence tags were obtained from each of the three S. aureus-infected libraries and the control library. Referenced to the control, 1720, 219, and 427 differentially expressed unique genes were identified in the pBMECs infected with S56, S178 and S36 S. aureus strains respectively. Gene ontology (GO and pathway analysis of the S56-infected pBMECs referenced to those of the control revealed that the differentially expressed genes in S56-infected pBMECs were significantly involved in inflammatory response, cell signalling pathways and apoptosis. In the same vein, the clustered GO terms of the differentially expressed genes of the S178-infected pBMECs were found to comprise immune responses, metabolism transformation, and apoptosis, while those of the S36-infected pBMECs were primarily involved in cell cycle progression and immune responses. Furthermore, fundamental differences were observed in the levels of expression of immune-related genes in response to treatments with the three S. aureus strains. These differences were especially noted for the expression of important pro-inflammatory molecules, including IL-1α, TNF, EFNB1, IL-8, and EGR1. The transcriptional changes associated with cellular signaling and the inflammatory response in this study may reflect different immunomodulatory mechanisms

  13. Enrichment for Repopulating Cells and Identification of Differentiation Markers in the Bovine Mammary Gland.

    Science.gov (United States)

    Rauner, Gat; Barash, Itamar

    2016-06-01

    Elucidating cell hierarchy in the mammary gland is fundamental for understanding the mechanisms governing its normal development and malignant transformation. There is relatively little information on cell hierarchy in the bovine mammary gland, despite its agricultural potential and relevance to breast cancer research. Challenges in bovine-to-mouse xenotransplantation and difficulties obtaining bovine-compatible antibodies hinder the study of mammary stem-cell dynamics in this species. In-vitro indications of distinct bovine mammary epithelial cell populations, sorted according to CD24 and CD49f expression, have been provided. Here, we successfully transplanted these bovine populations into the cleared fat pads of immunocompromised mice, providing in-vivo evidence for the multipotency and self-renewal capabilities of cells that are at the top of the cell hierarchy (termed mammary repopulating units). Additional outgrowths from transplantation, composed exclusively of myoepithelial cells, were indicative of unipotent basal stem cells or committed progenitors. Sorting luminal cells according to E-cadherin revealed three distinct populations: luminal progenitors, and early- and late-differentiating cells. Finally, miR-200c expression was negatively correlated with differentiation levels in both the luminal and basal branches of the bovine mammary cell hierarchy. Together, these experiments provide further evidence for the presence of a regenerative entity in the bovine mammary gland and for the multistage differentiation process within the luminal lineage. PMID:26615610

  14. Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice.

    Directory of Open Access Journals (Sweden)

    Gat Rauner

    Full Text Available Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland's epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse-bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology's development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor

  15. Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells

    Science.gov (United States)

    Xia, Xiaojing; Che, Yanyi; Gao, Yuanyuan; Zhao, Shuang; Ao, Changjin; Yang, Hongjian; Liu, Juxiong; Liu, Guowen; Han, Wenyu; Wang, Yuping; Lei, Liancheng

    2016-01-01

    During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma (IFN-γ) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether IFN-γ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether IFN-γ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of IFN-γ on milk synthesis in primary BMECs in vitro. The results showed that IFN-γ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following IFN-γ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that IFN-γ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor 2α (eIF2α) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate IFN-γ-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the IFN-γ-induced decrease in milk quality but also a useful therapeutic approach for IFN-γ-associated breast diseases in other animals and humans. PMID:27025389

  16. Oxytocin binding sites in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  17. In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow.

    Directory of Open Access Journals (Sweden)

    Han Hu

    Full Text Available BACKGROUND: The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. METHODOLOGY: Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. PRINCIPAL FINDINGS: The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n=60. Furthermore, they were capable of synthesizing beta-casein (CSN2, acetyl-CoA carboxylase-alpha (ACACA and butyrophilin (BTN1A1. An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. CONCLUSIONS: The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs.

  18. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

    Science.gov (United States)

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H.; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers. PMID:27028005

  19. Cell-cell interactions promote mammary epithelial cell differentiation

    OpenAIRE

    1985-01-01

    Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interac...

  20. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

    Science.gov (United States)

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment. PMID:27272504

  1. Specialized membrane biogenesis in mammary epithelial cells

    International Nuclear Information System (INIS)

    The apical membrane of the mammary gland epithelial cell is highly differentiated and adapted to participate in the process of fat secretion. Certain of the apical membrane differentiation antigens are frequently expressed on membrane carcinoma cells, and knowledge of the normal mechanisms by which these antigens are regulated may have implications for a better understanding of tumor antigen expression. Because the apical membrane of the cell is lost during secretion, active membrane biosynthesis must accompany fat secretion, and the cell represents a good model for studying membrane biogenesis in polarized epithelial cells. Experiments have been carried out using primary cultures of cells established from mammary glands of late pregnant mice and also a mouse cell line, COMMA-1-D, that differentiates in an appropriate milieu. When fat globule membranes are purified from mouse milk and the protein composition analyzed by SDS-polyacrylamide gel electrophoresis, four major proteins are identifiable with molecular weights of 55, 67, 90, and 150 kDa. The 67-kDa component was identified as butyrophilin and the 150-kDa one as xanthine oxidase. In addition, a high molecular weight carbohydrate rich glycoprotein, PAS-O, is also present. 3 refs., 3 figs

  2. Bovine mammary stem cells: Cell biology meets production agriculture

    Science.gov (United States)

    Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue ...

  3. Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens

    Czech Academy of Sciences Publication Activity Database

    Sorg, G.; Danowski, K.; Korenková, Vlasta; Rusňáková, Vendula; Kueffner, R.; Zimmer, R.; Meyer, H.H.D.; Kliem, H.

    2013-01-01

    Roč. 7, č. 5 (2013), s. 799-805. ISSN 1751-7311 Institutional research plan: CEZ:AV0Z50520701 Keywords : bovine mastitis * gene expression profiling * microfluidic qPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.784, year: 2013

  4. Spleen tyrosine kinase regulates mammary epithelial cell proliferation in mammary glands of dairy cows.

    Science.gov (United States)

    Hou, Xiaoming; Lin, Lin; Xing, Weinan; Yang, Yang; Duan, Xiaoyu; Li, Qingzhang; Gao, Xuejun; Lin, Ye

    2016-05-01

    Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows. PMID:26947307

  5. TCDD exposure disrupts mammary epithelial cell differentiation and function

    OpenAIRE

    Collins, Loretta L.; Lew, Betina J.; Lawrence, B. Paige

    2009-01-01

    Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced β-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less ...

  6. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  7. Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection

    Directory of Open Access Journals (Sweden)

    Vuocolo Tony

    2008-06-01

    Full Text Available Abstract Background Mastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection. Results Transcriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding β-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12 and Pentraxin-3 (PTX3 corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement

  8. Lactoferrin affects the adherence and invasion of Streptococcus dysgalactiae ssp. dysgalactiae in mammary epithelial cells.

    Science.gov (United States)

    O'Halloran, Fiona; Beecher, Christine; Chaurin, Valerie; Sweeney, Torres; Giblin, Linda

    2016-06-01

    Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence. PMID:27016824

  9. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    Full Text Available 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.

  10. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

    NARCIS (Netherlands)

    Lammers, A.; Vorstenbosch, van C.J.; Erkens, J.H.F.; Smith, H.E.

    2001-01-01

    We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover. we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other importan

  11. CIRCADIAN CLOCK AND CELL CYCLE GENE EXPRESSION: IN MOUSE MAMMARY EPITHELIAL CELLS AND IN THE DEVELOPING MOUSE MAMMARY GLAND

    OpenAIRE

    Metz, Richard P.; Qu, Xiaoyu; Laffin, Brian; Earnest, David; Porter, Weston W.

    2006-01-01

    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 ...

  12. Impact of let-7g on Proliferation and Lactation of Mouse Mammary Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Feng Li; Li Qing-zhang; Cui Wei; Ding Wei

    2012-01-01

    let-7g, a member of the let-7 family, regulates gene expression at the post-transcriptional level. The study explored a series of biological effects of mouse mammary epithelial cells that let-7g was produced. The differential expression of let-7g was detected by qRT-PCR in different developmental stages of the mouse mammary gland, let-7g expression and impact of let-7g on mouse mammary epithelial cells were analyzed by CASY-technology, qRT-PCR, Western blotting and HPLC inhibited let-7g expression of mouse mammary epithelial ceils through gene silencing. The results showed that qRT-PCR identified let-7g as being down-regulated in mouse mammary epithelial cells after it was inhibited. Mouse mammary epithelial cells with low expression of let-7g displayed higher expression of TGFβR I protein than those with high expression of let-7g, suggesting that low let-7g expression contributed to TGFβR I over-expression. Finally, the expression of let-7g was down-regulated, which significantly enhanced the proliferation of mouse mammary epithelial cells, and increased expression of β-Casein. The data indicated that let-7g could negatively regulate the expression of target Tgfbrl by complementary combination in mouse mammary epithelial cells, and then regulate the cell proliferation and expression of β-Casein by suppressing the TGFβR I expression.

  13. Binding of transcobalamin II by human mammary epithelial cells.

    Science.gov (United States)

    Adkins, Y; Lönnerdal, B

    2001-01-01

    The presence of nutrient binders in milk may have an important role during milk production and may influence the nutrient's bioavailability to the infant. Human milk and plasma contain at least two types of vitamin B12 binders: transcobalamin II (TCII) and haptocorrin (Hc). Vitamin B12 in milk is exclusively bound to Hc (Hc-B12). In plasma, the major vitamin B12 binding protein that is responsible for delivering absorbed vitamin B12 to most tissues and cells is TCII (TCII-B12). Currently, little is known about the route of secretion of vitamin B12 into human milk. It is possible that a receptor-mediated pathway is involved, since maternal vitamin B12 supplementation increases the amount of the vitamin secreted into human milk if the mother's vitamin B12 consumption is low, but remains unchanged if her intake is adequate. In this study, we investigated the process by which the mammary gland acquires vitamin B12 from maternal circulation, whether as a free vitamin or as a Hc-B12 or TCII-B12 complex. TCII was purified from plasma incubated with [57Co]vit B12 (B12*), while Hc was purified from whey incubated with B12*. Both proteins were separated by fast protein liquid chromatography using gel filtration and anion-exchange columns. Purity of the separated proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding studies were carried out on a monolayer of normal human mammary epithelial cells (HMEC) at 4 degrees C using free B12* and TCII-B12* and Hc-B12* complexes. Minimal binding of free B12* and Hc-B12* to HMEC was observed; however, HMEC exhibited a high affinity for the TCII-B12* complex. This study suggests that a specific cell surface receptor for the TCII-B12 complex exists in the mammary gland. It is possible that once vitamin B12 is in the mammary gland it is transferred to Hc (which may be synthesized by the mammary gland) and then secreted into milk as a Hc-B12 complex. PMID:11787717

  14. Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells.

    Science.gov (United States)

    Coloff, Jonathan L; Murphy, J Patrick; Braun, Craig R; Harris, Isaac S; Shelton, Laura M; Kami, Kenjiro; Gygi, Steven P; Selfors, Laura M; Brugge, Joan S

    2016-05-10

    Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation. PMID:27133130

  15. REGULATION OF GENE EXPRESSION IN THE BOVINE MAMMARY GLAND BY OVARIAN STEROIDS

    Science.gov (United States)

    It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent upon progesterone. Effects of these steroid hormones on gene expression in the mammary gland are ...

  16. Id-1 is not expressed in the luminal epithelial cells of mammary glands

    OpenAIRE

    Uehara, Norihisa; Chou, Yu-Chien; Galvez, Jose J; de-Candia, Paola; Cardiff, Robert D; Benezra, Robert; Shyamala, Gopalan

    2003-01-01

    Background The family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we exami...

  17. Paclitaxel resistance in untransformed human mammary epithelial cells is associated with an aneuploidy-prone phenotype

    OpenAIRE

    Bouchet, B P; Bertholon, J; Falette, N; Audoynaud, C; Lamblot, C; Puisieux, A; Galmarini, C M

    2007-01-01

    Despite its increasing clinical use, almost no data are currently available about paclitaxel effects on non-cancerous mammary epithelial cells. We have previously established paclitaxel-resistant sub-cell lines (paclitaxel-surviving populations, PSPs; n=20), and sensitive controls (control clones, CCs; n=10), from the untransformed human mammary epithelial cell line HME1. In this study, we aimed to establish whether paclitaxel resistance was associated with a modified sensitivity to paclitaxe...

  18. Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands.

    Directory of Open Access Journals (Sweden)

    Kentaro Nagaoka

    Full Text Available Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through lactation; its expression was stimulated by lactogenic hormone treatment of mammary epithelial cells. Lactogenic hormone also induced the expression of milk protein ß-casein mRNA (a marker of cell differentiation and E-cadherin mRNA (a marker of epithelial cells. However, knockdown of miR-200a prevented increases in ß-casein and E-cadherin mRNA expression. Protein analysis revealed that E-cadherin signal was decreased and ZEB1 (a marker of EMT was increased following miR-200a knockdown. Finally, in a three-dimensional culture system modeling lumen-containing mammary ducts, miR-200a knockdown decreased the cavity formation rate and suppressed claudin-3 and par-6b expression, indicating reduced epithelial cell polarity. These observations suggest that miR-200a is important for maintaining the epithelial cell phenotype, which contributes to lactogenic hormone induction of cellular differentiation in mammary glands.

  19. Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells.

    OpenAIRE

    Smith, G. H.; Gallahan, D; Zwiebel, J A; Freeman, S M; Bassin, R H; Callahan, R

    1991-01-01

    Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved...

  20. Epithelial Cell Differentiation Regulated by MicroRNA-200a in Mammary Glands

    OpenAIRE

    NAGAOKA, Kentaro; Zhang, Haolin; Watanabe, Gen; Taya, Kazuyoshi

    2013-01-01

    Mammary gland epithelial cells undergo periodic cycles of proliferation, differentiation, and involution. Many studies have reported that miRNAs, which are small, non-coding RNAs, influence a variety of biological processes during posttranscriptional regulation. Here, we found that one miRNA, miR-200a, was relatively highly expressed in epithelial cell-rich organs such as mammary glands, lung, and kidney in mice. In mammary glands, miR-200a expression increased during mid-pregnancy through la...

  1. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Mouse mammary epithelial cells (MMEC) secrete a group of milk-specific proteins including various caseins and whey proteins. Dissociated mammary epithelial cells maintain expression of most of their differentiated functions only if cells are plated on a suitable substratum. Casein production and section, cell morphology, and production of α-lactalbumin have been used as markers to assess the degree of differentiation of mammary cells in culture. The general consensus is that cells express their differentiated properties at high levels and for longer periods of time on such substrata. In this paper, the authors demonstrate that modulation of the expression of caseins by floating collagen gels is manifested at several regulatory points

  2. 茶皂素对奶牛乳腺上皮细胞增殖及乳脂合成关键酶的影响%Effects of Tea Saponin on Proliferation and Milk Fat Synthesis Key Enzymes of Bovine Mammary Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    严淑红; 邢文丽; 方洛云; 王俊杰; 刘续航; 蒋林树

    2015-01-01

    This experiment was conducted to study the effects of tea saponin on proliferation and milk fat syn⁃thesis key enzymes of bovine mammary epithelial cells. Mammary epithelial cells were obtained by the method of enzyme digestion. After immunofluorescence identification was done, mammary epithelial cells were cul⁃tured in tea saponin solution of different concentrations [0 (control), 0.05, 0.25, 0.50, 1.00, 5.00, 10.00, 20.00, 40.00, 60.00, 80.00 and 100.00 μg/mL]. 1) Thiazolyl blue tetrazolium bromide (MTT) method was used to detect the effects of tea saponin on the proliferation of cells. 2) Enzyme⁃linked immunosorbent as⁃say ( ELISA) kit was used to measure acetyl⁃CoA carboxylase ( ACACA) , fatty acid synthase ( FASN) and stearoyl⁃coenzyme A desaturase ( SCD ) contents in cells. 3 ) Fluorescence⁃based quantitative real⁃time PCR method was used to measure the relative expression level of mRNA of ACACA, FASN and SCD. The results showed as follows:1) compared with control group, there was no significant effect of tea saponin at concen⁃tration of 0.05 to 5.00 μg/mL ( P>0.05) , but the cell proliferation was significantly inhibited by tea saponin at concentration of 10.00 to 100.00 μg/mL ( P0.05); 3) compared with control group, the relative expression level of SCD mRNA in 0. 50, 5. 00 and 20.00 μg/mL concentration groups was significantly decreased ( P<0.05) . In conclusion, tea saponin plays a role in inhibition of proliferation and the relative expression level of fat synthesis key enzyme gene SCD mRNA of bovine mammary epithelial cells.%本试验旨在研究茶皂素对乳腺上皮细胞增殖、乳脂合成关键酶的影响。采用酶消化法分离获取乳腺上皮细胞,经免疫荧光鉴定后,分别添加不同浓度[0(对照)、0.05、0.25、0.50、1.00、5.00、10.00、20.00、40.00、60.00、80.00、100.00μg/mL]的茶皂素溶液培养,然后进行如下操作:1)采用噻唑蓝( MTT)法

  3. Id-1 is not expressed in the luminal epithelial cells of mammary glands

    International Nuclear Information System (INIS)

    The family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we examined its cellular localization in vivo using immunohistochemistry. Extracts of whole mammary glands from wild type and Id-1 null mutant mice, and tissue sections from paraffin-embedded mouse mammary glands from various developmental stages and normal human breast were subjected to immunoblot and immunohistochemical analyses, respectively. In both these procedures, an anti-Id-1 rabbit polyclonal antibody was used for detection of Id-1. In immunoblot analyses, using whole mammary gland extracts, Id-1 was detected. In immunohistochemical analyses, however, Id-1 was not detected in the luminal epithelial cells of mammary glands during any stage of development, but it was detected in vascular endothelial cells. Id-1 is not expressed in the luminal epithelial cells of mammary glands

  4. PUMA Cooperates with p21 to Regulate Mammary Epithelial Morphogenesis and Epithelial-To-Mesenchymal Transition.

    Directory of Open Access Journals (Sweden)

    Yanhong Zhang

    Full Text Available Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT.

  5. Effects of Chinese herbal medicine for warming kidney on proliferation of normal human mammary epithelial cells in primary culture

    OpenAIRE

    Ye, Mei-na; Chen, Hong-feng

    2006-01-01

    Objective: To evaluate the effects of Chinese herbal medicine for warming kidney on proliferation of normal human mammary epithelial cells in primary culture. Methods: The normal human mammary epithelial cells were dissociated by digestion with collagenase type Ⅰ. The morphological identification of normal human mammary epithelial cells in primary culture was determined under inverted phase contrast microscope and transmission electron microscope. The estradiol and progesterone were added to ...

  6. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  7. Radiation-induced chromosomal instability in human mammary epithelial cells

    Science.gov (United States)

    Durante, M.; Grossi, G. F.; Yang, T. C.

    1996-01-01

    Karyotypes of human cells surviving X- and alpha-irradiation have been studied. Human mammary epithelial cells of the immortal, non-tumorigenic cell line H184B5 F5-1 M/10 were irradiated and surviving clones isolated and expanded in culture. Cytogenetic analysis was performed using dedicated software with an image analyzer. We have found that both high- and low-LET radiation induced chromosomal instability in long-term cultures, but with different characteristics. Complex chromosomal rearrangements were observed after X-rays, while chromosome loss predominated after alpha-particles. Deletions were observed in both cases. In clones derived from cells exposed to alpha-particles, some cells showed extensive chromosome breaking and double minutes. Genomic instability was correlated to delayed reproductive death and neoplastic transformation. These results indicate that chromosomal instability is a radiation-quality-dependent effect which could determine late genetic effects, and should therefore be carefully considered in the evaluation of risk for space missions.

  8. Effect of ionizing radiation on nitric oxide production in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Full text: Nitric oxide (NO) is an important biological molecule with a wide variety of functions in physiological and pathophysiological events. We reported previously the presence of nitric oxide synthase (NOS) isoforms such as inducible, endothelial and neuronal types in the rat mammary glands. In addition, we demonstrated that a selective inhibitor of inducible NOS and NO-specific scavenger prevent radiation-induced rat mammary tumors, and that radiation-induced NO might contribute to tumor initiation of mammary glands in rat. However, the production and action of NO in the epithelium of mammary glands after the exposure of radiation are still unclear. In this current study we, therefore, examined the effects of ionizing radiation on a mouse mammary epithelial cell line (HC11) to provide a concrete evidence regarding the production of NO in the mammary epithelial cells after irradiation. The HC11 cells, established from COMMA-1D mouse mammary epithelial cell line, were cultured in RPMI-1640 growth medium containing 10% FCS, EGF and insulin until become confluence, then irradiated by X-ray with a dose at 10 or 30 Gy (1 Gy/min). After the irradiation, NO produced and secreted by HC11 cells into culture medium was estimated by the measurement of nitrite concentration in the culture medium with a Griess assay. HC11 cells produced NO spontaneously, and NO concentration was gradually increased during the experimented period. On the other hand, NO production was transiently enhanced immediately after irradiation of the cells in a dose-dependent manner. It, then, descended in an hour after irradiation, and returned to a basal level in 24 hours. These indicate that NO production was undoubtedly increased by irradiation in mammary epithelial cells, and support our previous propose that radiation-induced NO might contribute to initiation of tumorigenesis of mammary glands

  9. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Twite, Nicolas [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Andrei, Graciela [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Kummert, Caroline [ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Donner, Catherine [Department of Obstetrics and Gynecology, Erasme Hospital, Route de Lennik 808, 1070 Brussels (Belgium); Perez-Morga, David [Laboratory of Molecular Parasitology, Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, Gosselies (Belgium); De Vos, Rita [Pathology Department, U.Z. Leuven, Minderbroedersstraat 12, Leuven (Belgium); Snoeck, Robert, E-mail: Robert.Snoeck@Rega.kuleuven.be [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Marchant, Arnaud, E-mail: arnaud.marchant@ulb.ac.be [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium)

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  10. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    International Nuclear Information System (INIS)

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ

  11. Primary cilia distribution and orientation during involution of the bovine mammary gland.

    Science.gov (United States)

    Biet, J; Poole, C A; Stelwagen, K; Margerison, J K; Singh, K

    2016-05-01

    The regulation of mammary gland involution occurs through multiple levels including environmental factors, hormones, and local intramammary signals. Primary cilia (PC) are signaling organelles that sense biochemical and biophysical extracellular stimuli and are vital for cellular and tissue function. The aim of this study was to examine the distribution, incidence, and orientation of PC. Furthermore, we determined changes in expression levels of the signal transducer and activator of transcription (STAT)6 at the onset of bovine mammary gland involution. Mammary tissue was collected from pasture-fed, primiparous, nonpregnant Friesian dairy cows at mid lactation (n=5 per group) killed 6-h after milking (lactating controls) and during involution after 7 and 28 d of nonmilking (NM). Fluorescent immunohistochemistry and confocal microscopy of tissue sections showed that PC were present on luminal secretory epithelial cells (SEC), myoepithelial cells (MEC), and stromal fibroblast cells (SFC). Furthermore, in all 3 experimental groups, different PC positions or orientations relative to the cell surface were identified on SEC and MEC, which projected toward the lumen and were either straight, bent, or deflected against the apical cell surface, whereas PC in SFC were confined to the interalveolar space. However, by 28-d NM, fewer PC projected into the luminal space and most appeared deflected or projected toward the interalveolar space. Furthermore, by 28-d NM, with the increase in stromal connective tissue, more PC were detected within the interalveolar and interlobular stroma. At 28-d NM, we observed a decrease in luminal cilia relative to the total number of cilia. The number of ciliated cells in the total fraction (SEC, MEC, and SFC) was the same for all 3 groups, although in the luminal fraction (SEC and MEC), PC per nuclei increased by 28-d NM relative to lactation. At all 3 stages, we detected variations in shape and orientation of PC within the same alveolus, with

  12. Lipoprotein Lipase, Tissue Expression and Effects on Genes Related to Fatty Acid Synthesis in Goat Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Wang-Sheng Zhao

    2014-12-01

    Full Text Available Lipoprotein lipase (LPL serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC, the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively. Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis.

  13. Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism.

    Directory of Open Access Journals (Sweden)

    Bat-Chen Cohen

    Full Text Available Milk fat globule size is determined by the size of its precursors-intracellular lipid droplets-and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P 3 μm and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001. In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001. In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content.

  14. Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

    OpenAIRE

    1992-01-01

    Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine ...

  15. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization.

    Science.gov (United States)

    Fornetti, J; Flanders, K C; Henson, P M; Tan, A-C; Borges, V F; Schedin, P

    2016-02-01

    After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and

  16. Effects of N-acetylimidazole on oxytocin binding in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, X.; Gorewit, R.C.; Currie, W.B. (Cornell Univ., Ithaca NY (USA))

    1990-01-01

    The effects of N-acetylimidazole on specific binding of oxytocin to microsomal fractions of bovine mammary gland were studied. N-acetylimidazole suppressed oxytocin binding, with time and concentration dependence. Decreased oxytocin binding activity appeared to be due to decreased affinity of the hormone for its receptor. Acetylation of oxytocin, rather than of oxytocin receptors, seemed to be responsible for the decreased binding.

  17. Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation

    International Nuclear Information System (INIS)

    A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones ± fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable 35S-labelled proteins were monitored. Milk protein (αs,1-casein, lactoferrin (LF), α-lactalbumin, and β-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay

  18. Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

    OpenAIRE

    Nakles, Rebecca E.; Millman, Sarah L.; Cabrera, M. Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S.; Schroeder, Timm; Furth, Priscilla A.

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without...

  19. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

    Science.gov (United States)

    Ma, Li-bing; He, Xiao-ning; Si, Wan-tong; Zheng, Yue-Mao

    2016-01-01

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  20. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

    Science.gov (United States)

    He, Xiao-Ying; Ma, Li-Bing; He, Xiao-Ning; Si, Wan-Tong; Zheng, Yue-Mao

    2016-06-30

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  1. Image-based Evaluation of the Molecular Events Underlying HC11 Mammary Epithelial Cell Differentiation

    OpenAIRE

    Shan, Liang; Zhang, Renshu; Zhang, Wanghai; Lee, Edward; Sridhar, Rajagopalan; Snyderwine, Elizabeth G.; Wang, Paul C.

    2008-01-01

    We have developed an image-based technique for signal pathway analysis, target validation and compound screening related to mammary epithelial cell differentiation. This technique utilized the advantages of optical imaging and HC11-Lux model system. HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of β-casein gene promoter (p-344/-1βc-Lux). The promoter activity was imaged optically in real time following lactogenic induction. Th...

  2. The role of dietary selenium in bovine mammary gland health and immune function.

    Science.gov (United States)

    Salman, S; Khol-Parisini, A; Schafft, H; Lahrssen-Wiederholt, M; Hulan, H W; Dinse, D; Zentek, J

    2009-06-01

    Mastitis is not only a major cause of economic losses to the dairy industry but also a major problem in ensuring the quality and safety of the milk, associated with high somatic cell counts and residues of antibiotics used for treatment. One innovative approach to protection against mastitis is to stimulate the animal's natural defense mechanisms. Technological advances in immunological research have increased our ability to exploit the immunity of the bovine mammary gland during periods of high susceptibility to disease. The trace element selenium affects the innate and the adaptive immune responses of the mammary gland through cellular and humoral activities. Substantial research has been carried out on the effect of selenium (Se) on the immune function of the mammary gland and subsequent improvement in bovine udder health and mastitis control. Levels higher than current recommendations and Se-yeast can potentially be used to enhance our capacity to modulate the physiological mechanisms of the bovine mammary gland to respond to infection. This article provides an overview of the most recent research in this field. PMID:19195425

  3. Investigating the role of Integrin Linked Kinase in mammary epithelial cell differentiation

    OpenAIRE

    Rooney, Nicholas

    2014-01-01

    Epithelial cell adhesion to the surrounding extracellular matrix (ECM) is necessary for their proper behaviour and function. During pregnancy and lactation mammary epithelial cells (MECs) require signals imparted by specific β1 integrin-laminin interactions for their functional differentiation in response to Prolactin (Prl) and for the correct formation of polarised secretory acini. Downstream of β1 integrin (β1Itg), the scaffold protein Integrin Linked Kinase (ILK) has been identified as the...

  4. Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

    OpenAIRE

    Cortes, Diego F.; Sha, Wei; Hower, Valerie; Blekherman, Greg; Laubenbacher, Reinhard; Akman, Steven; Torti, Suzy V; Shulaev, Vladimir

    2011-01-01

    Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMEC), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5), were exposed to increased levels of rea...

  5. Embryonic stem cells are redirected to non-tumorigenic epithelial cell fate by interaction with the mammary microenvironment.

    Directory of Open Access Journals (Sweden)

    Corinne A Boulanger

    Full Text Available Experiments were conducted to redirect mouse Embryonic Stem (ES cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1:5 and 1:50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+ progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. We demonstrate that these progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas.

  6. Peripheral serotonin regulates maternal calcium trafficking in mammary epithelial cells during lactation in mice.

    Directory of Open Access Journals (Sweden)

    Jimena Laporta

    Full Text Available Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood into the ductal lumen (milk. Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1, which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2 and basolateral (CaSR, ORAI-1 membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2. Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2 are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation.

  7. Peripheral serotonin regulates maternal calcium trafficking in mammary epithelial cells during lactation in mice.

    Science.gov (United States)

    Laporta, Jimena; Keil, Kimberly P; Vezina, Chad M; Hernandez, Laura L

    2014-01-01

    Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation. PMID:25299122

  8. PUMA Cooperates with p21 to Regulate Mammary Epithelial Morphogenesis and Epithelial-To-Mesenchymal Transition

    OpenAIRE

    Zhang, Yanhong; Yan, Wensheng; Jung, Yong Sam; Chen, Xinbin

    2013-01-01

    Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multipl...

  9. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Simian, M.; Harail, Y.; Navre, M.; Werb, Z.; Lochter, A.; Bissell, M.J.

    2002-03-06

    The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP-3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a critical role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.

  10. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

    International Nuclear Information System (INIS)

    The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP-3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a critical role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland

  11. Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro.

    Directory of Open Access Journals (Sweden)

    Laia Hernández

    Full Text Available Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged-but not to young-human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies.

  12. Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

    OpenAIRE

    1987-01-01

    Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an underst...

  13. Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo.

    Science.gov (United States)

    Chen, Qiuming; Wu, Yanjun; Zhang, Mingyuan; Xu, Wenwen; Guo, Xiaoping; Yan, Xueyu; Deng, Haiying; Jiang, Qinyang; Yang, Xiurong; Lan, Ganqiu; Guo, Yafen; Qin, Guangsheng; Jiang, Hesheng

    2016-05-01

    Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells. PMID:27032540

  14. The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

    Science.gov (United States)

    Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

    2016-07-01

    Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (milk protein; milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. PMID:27132107

  15. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  16. Effects of β-Hydroxybutyric Acid on Relative Expression Levels of Genes Related to Milk Fat Synthesis in Bovine Mammary Epithelial Cells%β-羟丁酸对奶牛乳腺上皮细胞内乳脂肪合成及其相关基因相对表达量的影响

    Institute of Scientific and Technical Information of China (English)

    常晨城; 齐利枝; 闫素梅; 生冉; 赵艳丽

    2015-01-01

    本试验主要研究了不同浓度的β-羟丁酸( BHBA)对奶牛乳腺上皮细胞( BMECs)活力、甘油三酯( TAG)含量、脂滴形成以及乳脂肪合成相关基因转录水平的影响。将传至第3代的BMECs悬液(1×105个/孔)接种于细胞培养板上,每孔加入含10%胎牛血清( FBS)的DMEM/F12培养液,于37℃的5%二氧化碳(CO2)培养箱培养48 h。再将培养48 h的BMECs随机分配到6个组,各组向培养孔中加入含不同浓度BHBA的DMEM/F12培养液,培养液中的FBS用1 g/L无脂肪酸的牛血清白蛋白( BSA)代替,并使反应体系中BHBA的最终浓度分别为0(对照)、0.58、1.16、2.32、4.64和9.28 mmol/L。置于37℃的5%CO2培养箱继续培养48 h。试验结果显示:随着BHBA浓度的增加,BMECs活力[(相对增殖率( RGR)]呈显著的二次曲线增加(P=0.041),其中BMECs活力以0.58~4.64 mmol/L BHBA组较高,9.28 mmol/L BHBA组较低;低浓度(0.58~2.32 mmol/L)的 BHBA 可促进 BMECs 内脂滴的形成,而较高浓度(4.64~9.28 mmol/L)的BHBA对脂滴形成的促进作用减弱;BHBA与TAG含量及乳脂肪合成相关基因脂肪酸合成酶( FASN )、乙酰辅酶 A 羧化酶α( ACACA )、硬脂酰辅酶 A 去饱和酶( SCD)、脂肪酸结合蛋白3( FABP3)、过氧化物酶体增殖物激活受体γ( PPARG)和分化抗原簇36(CD36)的相对表达量均无显著的一次线性或二次曲线关系(P>0.05)。综上,BHBA 对BMECs活力的促进作用呈显著的二次曲线增加,即BHBA对BMECs活力呈显著浓度依赖关系;BHBA对细胞内乳脂肪的合成有提高的趋势。%This study was conducted to determine the effects ofβ-hydroxybutyric acid ( BHBA) on cell viabili-ty, triacylglycerol ( TAG) content, lipid droplet formation and relative expression levels of genes related to milk fat synthesis in bovine mammary epithelial cells (BMECs). The 3th passage cells were plated 1×105 cells/well in culture plates and DMEM/F12 medium containing 10% fetal bovine serum ( FBS) was added to

  17. Role of endothelial cells in bovine mammary gland health and disease.

    Science.gov (United States)

    Ryman, Valerie E; Packiriswamy, Nandakumar; Sordillo, Lorraine M

    2015-12-01

    The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood-milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium. PMID:26303748

  18. Adipose triglyceride lipase regulates lipid metabolism in dairy goat mammary epithelial cells.

    Science.gov (United States)

    Li, Jun; Luo, Jun; Wang, Hui; Shi, Hengbo; Zhu, Jiangjiang; Sun, Yuting; Yu, Kang; Yao, Dawei

    2015-01-01

    Adipose triglyceride lipase (ATGL) catalyzes the initial step in the lipid lipolysis process, hydrolyzing triglyceride (TG) to produce diacylglycerol (DG) and free fatty acids (FFA). In addition, ATGL regulates lipid storage and release in adipocyte cells. However, its role in mammary gland tissue remains unclear. To assess the role of the ATGL gene in the goat mammary gland, this study analyzed the tissue distribution and expression of key genes together with lipid accumulation after knockdown of the ATGL gene. The mRNA of ATGL was highly expressed in subcutaneous adipose tissue, the lung and the mammary gland with a significant increase in expression during the lactation period compared with the dry period of the mammary gland. Knockdown of the ATGL gene in goat mammary epithelial cells (GMECs) using siRNA resulted in a significant decrease in both ATGL mRNA and protein levels. Silencing of the ATGL gene markedly increased lipid droplet accumulation and intracellular TG concentration (Pfat formation and secretion was down-regulated (PCD36 for fatty acid uptake (P<0.05). In conclusion, these data suggest that the ATGL gene plays an important role in triglyceride lipolysis in GMECs and provides the first experimental evidence that ATGL may be involved in lipid metabolism during lactation. PMID:25307872

  19. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally

    OpenAIRE

    1989-01-01

    We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM- 2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monocult...

  20. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures

    Directory of Open Access Journals (Sweden)

    Mainil Jacques

    2005-12-01

    Full Text Available Abstract Background Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. Results Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein as well as an epithelial cytoskeleton component (cytokeratin 18. However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker. Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1–2 mM or using a glucose-deprived culture medium. Conclusion The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2002-02-22

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

  3. Trichostatin A Inhibits β-Casein Expression in Mammary Epithelial Cells

    Science.gov (United States)

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2010-01-01

    Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein β-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous β-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of β-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types. PMID:11746508

  4. Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

    Directory of Open Access Journals (Sweden)

    Earp H Shelton

    2010-12-01

    Full Text Available Abstract Background Mammary glands harbor a profound burden of apoptotic cells (ACs during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis. Results ACs induced MerTK expression in MECs, resulting in elevated MerTK levels at the earliest stages of involution. Loss of MerTK resulted in AC accumulation in post-lactational MerTK-deficient mammary glands, but not in Axl and Tyro3-deficient mammary glands. Increased vascularization, fibrosis, and epithelial hyperproliferation were observed in MerTK-deficient mammary glands through at least 60 days post-weaning, due to failed efferocytosis after lactation, but did not manifest in nulliparous mice. WT host-derived macrophages failed to rescue efferocytosis in transplanted MerTK-deficient mammary epithelium. Conclusion Efferocytosis by MECs through MerTK is crucial for mammary gland homeostasis and function during the post-lactational period. Efferocytosis by MECs thus limits pathologic consequences associated with the apoptotic load following lactation.

  5. Metabolic history impacts mammary tumor epithelial hierarchy and early drug response in mice.

    Science.gov (United States)

    Montales, Maria Theresa E; Melnyk, Stepan B; Liu, Shi J; Simmen, Frank A; Liu, Y Lucy; Simmen, Rosalia C M

    2016-09-01

    The emerging links between breast cancer and metabolic dysfunctions brought forth by the obesity pandemic predict a disproportionate early disease onset in successive generations. Moreover, sensitivity to chemotherapeutic agents may be influenced by the patient's metabolic status that affects the disease outcome. Maternal metabolic stress as a determinant of drug response in progeny is not well defined. Here, we evaluated mammary tumor response to doxorubicin in female mouse mammary tumor virus-Wnt1 transgenic offspring exposed to a metabolically compromised environment imposed by maternal high-fat diet. Control progeny were from dams consuming diets with regular fat content. Maternal high-fat diet exposure increased tumor incidence and reduced tumor latency but did not affect tumor volume response to doxorubicin, compared with control diet exposure. However, doxorubicin-treated tumors from high-fat-diet-exposed offspring demonstrated higher proliferation status (Ki-67), mammary stem cell-associated gene expression (Notch1, Aldh1) and basal stem cell-like (CD29(hi)CD24(+)) epithelial subpopulation frequencies, than tumors from control diet progeny. Notably, all epithelial subpopulations (CD29(hi)CD24(+), CD29(lo)CD24(+), CD29(hi)CD24(+)Thy1(+)) in tumors from high-fat-diet-exposed offspring were refractory to doxorubicin. Further, sera from high-fat-diet-exposed offspring promoted sphere formation of mouse mammary tumor epithelial cells and of human MCF7 cells. Untargeted metabolomics analyses identified higher levels of kynurenine and 2-hydroxyglutarate in plasma of high-fat diet than control diet offspring. Kynurenine/doxorubicin co-treatment of MCF7 cells enhanced the ability to form mammosphere and decreased apoptosis, relative to doxorubicin-only-treated cells. Maternal metabolic dysfunctions during pregnancy and lactation may be targeted to reduce breast cancer risk and improve early drug response in progeny, and may inform clinical management of disease

  6. Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

    OpenAIRE

    Silim, A.; Elazhary, M. A. S. Y.

    1983-01-01

    Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell culture...

  7. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    International Nuclear Information System (INIS)

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with [3H]arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. [3H]arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein

  8. Regulation of functional cytodifferentiation and histogenesis in mammary epithelial cells: Role of the extracellular matrix

    International Nuclear Information System (INIS)

    Primary mammary epithelial cells provide a versatile system for the study of hormone and extracellular matrix (ECM) influences on tissue-specific gene expression. The authors have characterized the formation of aveolarlike morphogenesis and mammary-specific functional differentiation that occur when these cells are cultured on a reconstituted basement membrane (EHS). Cells cultured on EHS exhibit many ultrastructural and biochemical features indicative of polarized and functionally differentiated mammary epithelium in vivo. The increased expression and specific vectorial secretion of milk proteins into lumina formed in culture are accompanied by large increases in milk protein mRNA expression. However, when individual ECM components are tested, smaller increases in milk protein mRNA are measured on heparan sulfate proteoglycan (HSPG) and laminin, and these responses are not associated with full functional cytodifferentiation or histotypic configuration. This indicates that multiple levels of regulation are involved in mammary-specific gene expression, and that in addition to individual ligand requirements cooperative interactions between various ECM molecules and cells are necessary for functional differentiation in culture. They have also shown that endogenous production of ECM molecules and changes in cell geometry are correlated with changes in functional and histogenic gene expression. They have previously proposed a model of cell-ECM interactions that is consistent with these data

  9. Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chanson, L. [Ecole Polytechnique Federale de Lausanne (Switzerland). Inst. of Bioengineering; Brownfield, D. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Garbe, J. C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Kuhn, I. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Stampfer, M. R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Bissell, M. J. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; LaBarge, M. A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.

    2011-02-07

    Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.

  10. Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

    OpenAIRE

    Schmidhauser, C; Bissell, M.J.; Myers, C A; Casperson, G F

    1990-01-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the c...

  11. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations That Leads to Stable Epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    OpenAIRE

    Lochter, André; Galosy, Sybille; Muschler, John; Freedman, Neal; Werb, Zena; Bissell, Mina J.

    1997-01-01

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell–cell contact...

  12. Paradoxical antiproliferative effect by a murine mammary tumor-derived epithelial cell line

    International Nuclear Information System (INIS)

    Despite significant advancement in breast cancer therapy, there is a great need for a better understanding of the mechanisms involved in breast carcinogenesis and progression, as well as of the role of epigenetic contributions from stromal cells in mammary tumorigenesis. In this study, we isolated and characterized murine mammary tumor-derived epithelial and myofibroblast cell lines, and investigated the in vitro and in vivo effect of cellular soluble factors produced by the epithelial cell line on tumor cells. Morphology, immunophenotype, cytogenetics, invasiveness, and tumorigenicity of epithelial (LM-234ep) and myofibroblast (LM-234mf) cell lines isolated from two murine mammary adenocarcinomas with common ancestor were studied. The in vitro effects of LM-234ep conditioned medium on proliferation, cell cycle distribution, and expression of cell cycle proteins, were investigated in LM-234mf cells, mouse melanoma cells (B16-F10), and human cervical adenocarcinoma cells (HeLa). The in vivo anti-tumor activity of LM-234ep conditioned media was evaluated in subcutaneous tumors formed in nude mice by B16-F10 and HeLa cells. LM-234ep cells were found to be cytokeratin positive and hipertriploid, whereas LM-234mf cells were α-smooth muscle actin positive and hypohexaploid. Chromosome aberrations were found in both cases. Only LM-234mf revealed to be invasive in vitro and to secrete active MMP-2, though neither of the cell types were able to produce progressing tumors. LM-234ep-derived factors were able to inhibit the in vitro growth of LM-234mf, B16-F10, and HeLa cells, inducing cell cycle arrest in G0/G1 phase. The administration of LM-234ep conditioned medium inhibited the growth of B16-F10 and HeLa tumors in nude mice. Our data suggest the existence of epithelial cell variants with tumor suppressive properties within mammary tumors. To our knowledge, this is the first report showing antiproliferative and antineoplastic activities induced by tumor-derived epithelial

  13. Paracrine-acting adiponectin promotes mammary epithelial differentiation and synergizes with genistein to enhance transcriptional response to estrogen receptor beta signaling

    Science.gov (United States)

    Mammary stromal adipocytes constitute an active site for the synthesis of the adipokine adiponectin (APN) that may influence the mammary epithelial microenvironment. The relationship between 'local', mammary tissue-derived APN and breast cancer risk is poorly understood. Herein, we identify a novel ...

  14. Transcriptomic response of goat mammary epithelial cells to Mycoplasma agalactiae challenge – a preliminary study

    DEFF Research Database (Denmark)

    Ogorevc, Jernej; Mihevc, Sonja Prpar; Hedegaard, Jakob;

    2015-01-01

    , steroid metabolism, fatty acid metabolism, apoptosis signalling, transcription regulation, and cell cycle regulation. Based on the results we suggest that mammary epithelial cells in vivo contribute to the immune system by the induced expression of cytokines and other chemotactic agents, activation of the...... challenged with Ma. High-throughput mRNA sequencing was performed to reveal differentially expressed genes (DEG) at different time-points (3 h, 12 h, and 24 h) post infection (PI). The pathway enrichment analysis of the DEG showed that infection significantly affected pathways associated with immune response...... complement system and apoptosis pathways, and expression of genes coding for antimicrobial molecules and peptides. In our study we attempted to interpret the detected transcriptomic changes in a biological context and infer mammary infection resistance candidate genes, interesting for further validation...

  15. Age-Related Dysfunction in Mechanotransduction Impairs Differentiation of Human Mammary Epithelial Progenitors

    Directory of Open Access Journals (Sweden)

    Fanny A. Pelissier

    2014-06-01

    Full Text Available Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged 55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity.

  16. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  17. Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Ying Qu

    Full Text Available Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture, three-dimensional (3D "on-top" Matrigel, 3D "cell-embedded" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

  18. The bone morphogenetic protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro

    OpenAIRE

    Perotti, C.; Karayazi, Ö.; Moffat, S.; Shemanko, C. S.

    2012-01-01

    Bone morphogenetic proteins (BMPs) have been implicated in the control of proliferation, tissue formation, and differentiation. BMPs regulate the biology of stem and progenitor cells and can promote cellular differentiation, depending on the cell type and context. Although the BMP pathway is known to be involved in early embryonic development of the mammary gland via mesenchymal cells, its role in later epithelial cellular differentiation has not been examined. The majority of the mammary gla...

  19. Homotypic RANK signaling differentially regulates proliferation, motility and cell survival in osteosarcoma and mammary epithelial cells.

    Science.gov (United States)

    Beristain, Alexander G; Narala, Swami R; Di Grappa, Marco A; Khokha, Rama

    2012-02-15

    RANKL (receptor activator of NF-κB ligand) is a crucial cytokine for regulating diverse biological systems such as innate immunity, bone homeostasis and mammary gland differentiation, operating through activation of its cognate receptor RANK. In these normal physiological processes, RANKL signals through paracrine and/or heterotypic mechanisms where its expression and function is tightly controlled. Numerous pathologies involve RANKL deregulation, such as bone loss, inflammatory diseases and cancer, and aberrant RANK expression has been reported in bone cancer. Here, we investigated the significance of RANK in tumor cells with a particular emphasis on homotypic signaling. We selected RANK-positive mouse osteosarcoma and RANK-negative preosteoblastic MC3T3-E1 cells and subjected them to loss- and gain-of-RANK function analyses. By examining a spectrum of tumorigenic properties, we demonstrate that RANK homotypic signaling has a negligible effect on cell proliferation, but promotes cell motility and anchorage-independent growth of osteosarcoma cells and preosteoblasts. By contrast, establishment of RANK signaling in non-tumorigenic mammary epithelial NMuMG cells promotes their proliferation and anchorage-independent growth, but not motility. Furthermore, RANK activation initiates multiple signaling pathways beyond its canonical target, NF-κB. Among these, biochemical inhibition reveals that Erk1/2 is dominant and crucial for the promotion of anchorage-independent survival and invasion of osteoblastic cells, as well as the proliferation of mammary epithelial cells. Thus, RANK signaling functionally contributes to key tumorigenic properties through a cell-autonomous homotypic mechanism. These data also identify the likely inherent differences between epithelial and mesenchymal cell responsiveness to RANK activation. PMID:22421365

  20. 乙酸钠和β-羟丁酸钠对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响%Effects of Sodium Acetate and Sodium β-Hydroxybutyrate on ExPressions of Genes Involved in Milk Fat and Protein Synthesis in Bovine Mammary EPithelial Cells

    Institute of Scientific and Technical Information of China (English)

    塔娜; 李红磊; 侯先志; 考桂兰; 高民; 李大彪

    2014-01-01

    本试验旨在研究乙酸钠和β-羟丁酸钠对奶牛乳腺上皮细胞(BMECs)乳脂和乳蛋白合成相关基因表达的影响。试验分2部分,均采用单因子完全随机试验设计。第1部分,单独添加试验,乙酸钠的添加浓度分别为0(对照)、6.00、9.00、12.00和15.00 mmol/ L,β-羟丁酸钠的添加浓度分别为0(对照)、0.80、1.60和2.40 mmol/ L。第2部分,混合添加试验,以单独添加试验得出的乙酸钠和β-羟丁酸钠的适宜浓度,二者之和为总添加浓度,设定3种不同配比,即乙酸钠∶β-羟丁酸钠分别为1∶1、2∶1和4∶1,对照组不添加乙酸钠和β-羟丁酸钠。结果表明:1)与对照组相比,12.00 mmol/ L 乙酸钠能够显著提高 BMECs 乙酰辅酶 A 羧化酶( ACC)、脂肪酸合成酶(FAS)、二酰甘油酰基转移酶( DGAT)、乙酰辅酶 A 合成酶2( ACSS2)、过氧化物酶体增殖物激活受体(PPARG)、κ酪蛋白(CSN3)和雷帕霉素靶蛋白( mTOR)基因的表达量及甘油三酯(TAG)的含量( P <0.05)。2)与对照组相比,2.40 mmol/ L 的β-羟丁酸钠能够显著提高BMECs ACC、FAS、ACSS2、PPARG、mTOR 基因表达量及 TAG 含量(P<0.05)。3)添加不同配比的乙酸钠和β-羟丁酸钠均不同程度地促进了乳脂合成相关基因的表达,乙酸钠∶β-羟丁酸钠为2∶1和4∶1时,BMECs 中 TAG 含量显著高于为1∶1时和对照组( P<0.05)。综合各项指标,以9.60 mmol/ L乙酸钠和4.80 mmol/ L β-羟丁酸钠混合添加对奶牛乳腺上皮细胞乳脂和乳蛋白合成的促进效果较好。%The aim of this study was to determine the effects of sodium acetate and sodium β-hydroxybutyrate on expressions of genes involved in milk fat and protein synthesis in bovine mammary epithelial cells (BMECs). The study was consisted of two parts,and completely random single-factor designs were adopted. Part

  1. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.

    OpenAIRE

    Wazer, D E; X. L. Liu; Chu, Q.; Gao, Q.; Band, V

    1995-01-01

    Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predom...

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

    OpenAIRE

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and ar...

  3. Transcriptome MicroRNA Profiling of Bovine Mammary Glands Infected with Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Rui Li

    2015-03-01

    Full Text Available MicroRNAs are small non-coding RNA molecules that are important regulators of gene expression at the post-transcriptional level. miRNAs impact the processes of cell proliferation, differentiation and apoptosis. Thus, the regulation of miRNA expression profiles associated with mastitis will be conducive for its control. In this study, Staphylococcus aureus (S. aureus was administered to the mammary gland of Chinese Holstein cows to construct a bacteria-type mastitis model. Total RNA was isolated from bovine mammary gland tissue samples from the S. aureus-induced mastitis group and controls. miRNAs were analyzed using Solexa sequencing and bioinformatics processing for the experimental group and control group. Two miRNA libraries were constructed respectively. A total of 370 known bovine miRNAs and 341 novel mi RNAs were detected for the S. aureus and 358 known bovine miRNAs and 232 novel miRNAs for control groups. A total of 77 miRNAs in the S. aureus group showed significant differences compared to the control group. GO (Gene Ontology analysis showed these target genes were involved in the regulation of cells, binding, etc., while KEGG (Kyoto Encyclopedia of Genes and Genomes analysis showed that these genes were enriched in endocytosis, and olfactory transduction pathways involved in cancer. These results provide an experimental basis to reveal the cause and regulatory mechanism of mastitis and also suggest the potential of miRNAs to serve as biomarkers for the diagnosis of mastitis in dairy cows.

  4. Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Martina A Gärtner

    Full Text Available Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy bovine uteri and further characterised. Bovine endometrial epithelial cells in the second passage (n = 5 animals were co-cultured with the autochthonous isolates L. buchneri, L. ruminis and L. amylovorus as well as with a commercially available L. vaginalis in different multiplicities of infection (MOI = 1, 5 and 10, respectively. Endometrial epithelial cells cultured without bacteria served as controls. At distinct points in time (2, 4 and 6 h total RNA was extracted from co-cultured epithelial cells and subjected to reverse transcription quantitative PCR of pro-inflammatory factors. Furthermore, the release of such factors by co-cultured epithelial cells was measured by ELISA or EIA after 24 and 48 h. L. ruminis and L. amylovorus induced increased interleukin (IL IL1A, IL6, IL8 and prostaglandin-endoperoxide synthase 2 mRNA levels and the release of IL8 and prostaglandin F(2α in endometrial epithelial cells compared with control cells. In contrast, L. buchneri did not significantly influence the expression and release of these factors. Toll-like receptors 2 and 6 transcripts were found unchanged in co-cultured and untreated epithelial cells in vitro. However, endometrial epithelial cells of each animal showed individual differences in the response to bacterial load. These results suggest that Lactobacillus species are present in the bovine uterus, revealing immunomodulatory properties.

  5. Detection and characterisation of Lactobacillus spp. in the bovine uterus and their influence on bovine endometrial epithelial cells in vitro.

    Science.gov (United States)

    Gärtner, Martina A; Bondzio, Angelika; Braun, Nicole; Jung, Markus; Einspanier, Ralf; Gabler, Christoph

    2015-01-01

    Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy bovine uteri and further characterised. Bovine endometrial epithelial cells in the second passage (n = 5 animals) were co-cultured with the autochthonous isolates L. buchneri, L. ruminis and L. amylovorus as well as with a commercially available L. vaginalis in different multiplicities of infection (MOI = 1, 5 and 10, respectively). Endometrial epithelial cells cultured without bacteria served as controls. At distinct points in time (2, 4 and 6 h) total RNA was extracted from co-cultured epithelial cells and subjected to reverse transcription quantitative PCR of pro-inflammatory factors. Furthermore, the release of such factors by co-cultured epithelial cells was measured by ELISA or EIA after 24 and 48 h. L. ruminis and L. amylovorus induced increased interleukin (IL) IL1A, IL6, IL8 and prostaglandin-endoperoxide synthase 2 mRNA levels and the release of IL8 and prostaglandin F(2α) in endometrial epithelial cells compared with control cells. In contrast, L. buchneri did not significantly influence the expression and release of these factors. Toll-like receptors 2 and 6 transcripts were found unchanged in co-cultured and untreated epithelial cells in vitro. However, endometrial epithelial cells of each animal showed individual differences in the response to bacterial load. These results suggest that Lactobacillus species are present in the bovine uterus, revealing immunomodulatory properties. PMID:25803719

  6. Selective release of microRNA species from normal and malignant mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lucy Pigati

    Full Text Available MicroRNAs (miRNAs in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

  7. Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

    DEFF Research Database (Denmark)

    Mani, O; Sørensen, M T; Sejrsen, K; Bruckmaier, R M; Albrecht, C

    The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland...... functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these...... transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis....

  8. Bovine mammary dendritic cells: a heterogeneous population, distinct from macrophages and similar in phenotype to afferent lymph veiled cells.

    Science.gov (United States)

    Maxymiv, Nicolas G; Bharathan, Mini; Mullarky, Isis K

    2012-01-01

    Dendritic cells (DC) are a heterogeneous population of professional antigen presenting cells and are potent stimulators of naïve T-cells. However, there is little previous research describing DC in bovine mammary tissue, primarily because of the difficulty distinguishing these cells from macrophages, which possess a similar phenotype. Using immunohistofluorescence and a combination of markers (MHC-II, CD205, CD11c), DC were localized in the bovine mammary gland and supramammary lymph node. In mammary tissue DC were found within the alveolar epithelium and within the intralobular connective tissue. In the lymph node DC were found on the periphery of B-cell areas, in the cortex, and among T-cells in the paracortex and medulla. DC in mammary parenchyma and supramammary lymph nodes were quantified and further characterized using flow cytometry. DC were CD11c(hi), CD14(lo) cells that expressed MHC-II and CD205. DC could be distinguished from macrophages based on their low CD14 expression. This research provides a better understanding of mammary gland immunology, while potentially aiding in the targeting of antigens to mucosal DC for vaccine development. PMID:22019401

  9. Detection and Characterisation of Lactobacillus spp. in the Bovine Uterus and Their Influence on Bovine Endometrial Epithelial Cells In Vitro

    OpenAIRE

    Gärtner, Martina A.; Bondzio, Angelika; Braun, Nicole; Jung, Markus; Einspanier, Ralf; Gabler, Christoph

    2015-01-01

    Bacterial infections and inflammation of the uterus are common in dairy cattle after parturition. In particular, pathogenic bacteria that cause endometritis have been the focus of research in cattle reproduction in the last ten years. The aim of the present study was to identify commensal lactobacilli in the bovine uterus and to examine their influence on the synthesis of pro-inflammatory factors in bovine endometrial epithelial cells in vitro. Lactobacillus species were isolated from healthy...

  10. Proliferation of Estrogen Receptor alpha Positive Mammary Epithelial Cells is Restrained by TGFbeta1 in Adult Mice

    Energy Technology Data Exchange (ETDEWEB)

    Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.; Shyamala, G.; Moses, Harold L.; Barcellos-Hoff, Mary Helen

    2005-03-03

    Transforming growth factor {beta}1 (TGF{beta}1) is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor {alpha} (ER{alpha}) cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF{beta}1 is necessary for the quiescence of ER{alpha}-positive population, we examined mouse mammary epithelial gland at estrus. Approximately 35% of cells showed TGF{beta}1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF{beta} signaling is autocrine. Furthermore, nuclear Smad co-localized with nuclear ER{alpha}. To test whether TGF{beta} was functional, we examined genetically engineered mice with different levels of TGF{beta}1. ER{alpha} co-localization with markers of proliferation (i.e. Ki-67 or BrdU) at estrus was significantly increased in the mammary glands of Tgf{beta}1 C57/bl/129SV heterozygote mice. This relationship was maintained following pregnancy, but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF{beta}1 via the MMTV promoter suppressed proliferation of ER{alpha} positive cells. Thus, TGF{beta}1 activation functionally restrains ER{alpha} positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF{beta}1 dysregulation may promote proliferation of ER{alpha} positive cells associated with breast cancer risk in humans.

  11. Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

    Science.gov (United States)

    Nakles, Rebecca E; Millman, Sarah L; Cabrera, M Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S; Schroeder, Timm; Furth, Priscilla A

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions. PMID:23425702

  12. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    International Nuclear Information System (INIS)

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV

  13. Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line.

    OpenAIRE

    Ball, R K; Friis, R R; Schoenenberger, C A; Doppler, W; Groner, B

    1988-01-01

    In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin-responsive cell clone, HC11, from the COMMA-1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co-cultivation with other cell types for the prolactin-dependent in vitro induction of the endogenous beta-casein gene by lactogenic ...

  14. Enhanced growth medium and method for culturing human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R. (7290 Sayre Dr., Oakland, CA 94611); Smith, Helene S. (5693 Cabot Dr., Oakland, CA 94611); Hackett, Adeline J. (82 Evergreen Dr., Orinda, CA 94563)

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  15. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  16. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    International Nuclear Information System (INIS)

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.

  17. Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line.

    Science.gov (United States)

    Kaushik, Ramakant; Singh, Karn Pratap; Kumari, Archana; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2014-09-01

    The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 10(6) cells. PMID:24969480

  18. Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

    Directory of Open Access Journals (Sweden)

    Helbling Jean-Christophe

    2010-12-01

    Full Text Available Abstract Background Laser-capture microdissection (LCM that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR, microarrays and most recently by RNA-sequencing. Challenges are i to select precisely and efficiently cells of interest and ii to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC are predominant. However several other cell types, including myoepithelial (MMC and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems® system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity. Results The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems® and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini

  19. Mammary Carcinogenesis Is Preceded by Altered Epithelial Cell Turnover in Transforming Growth Factor-α and c-myc Transgenic Mice

    OpenAIRE

    Rose-Hellekant, Teresa A; Wentworth, Kristin M.; Nikolai, Sarah; Kundel, Donald W.; Sandgren, Eric P.

    2006-01-01

    Identification of biomarkers that indicate an increased risk of breast cancer or that can be used as surrogates for evaluating treatment efficacy is paramount to successful disease prevention and intervention. An ideal biomarker would be identifiable before lesion development. To test the hypothesis that changes in cell turnover precede mammary carcinogenesis, we evaluated epithelial cell proliferation and apoptosis in mammary glands from transgenic mice engineered to develop mammary cancer d...

  20. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

    Science.gov (United States)

    Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

    2001-01-01

    Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

  1. Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells

    OpenAIRE

    2010-01-01

    Background IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO), an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. Methods SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET) for dif...

  2. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Miura, Yuka; Hagiwara, Natsumi [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan); Radisky, Derek C. [Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32225 (United States); Hirai, Yohei, E-mail: y-hirai@kwansei.ac.jp [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan)

    2014-09-10

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.

  3. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination

  4. EGF stimulates Mg{sup 2+} influx in mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Trapani, Valentina; Arduini, Daniela; Luongo, Francesca; Wolf, Federica I., E-mail: federica.wolf@rm.unicatt.it

    2014-11-28

    Highlights: • EGF stimulation potentiates Mg{sup 2+} influx into epithelial cells. • EGF-induced Mg{sup 2+} influx does not depend on the concomitantly induced Ca{sup 2+} signal. • EGF-induced Ca{sup 2+} signal is dependent on the presence of extracellular Mg{sup 2+}. • New players in EGF-mediated signaling might be exploited as therapeutic targets. - Abstract: Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg{sup 2+} and Ca{sup 2+} fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg{sup 2+}, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg{sup 2+} derives from an influx from the extracellular compartment, and does not depend on Ca{sup 2+}. On the contrary, the increase in intracellular Ca{sup 2+} derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg{sup 2+} and Ca{sup 2+} signals. These findings demonstrate that not only does Mg{sup 2+} influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg{sup 2+} influx is essential for the Ca{sup 2+} signal to occur.

  5. Purification and characterization of glycoprotein processing enzyme α-glucosidase II from bovine mammary tissue

    International Nuclear Information System (INIS)

    α-Glucosidase II removes the two inner (α1-3) linked glucose residues of glycoprotein precursor Glc3Man9(GlcNAc)2.α-Glucosidase II has been purified to homogeneity from bovine mammary tissue. Briefly, purification involved solubilization of glucosidase II with Triton X-100 from microsomes followed by 20-60% (NH4)2SO4 fractionation, Con-A Sepharose 4B, hydroxylapatite and DEAE-Sephacel column chromatography. The purified glucosidase II showed single band on 10% non-denaturing PAGE and enzyme activity band on visualization with fluorogenic substrate 4-methylumbelliferyl (MUF) α-D-glucopyranoside. The purified enzyme exhibited broad pH optima (6.0-7.5). The enzyme hydrolyzed MUFα-D-glucoside (K/sub m/ = 13 μM) but was inactive against the corresponding D-glucoside and the α-D-mannoside. Cleavage of MUF α-glucoside was enhanced by mannose and starch and was inhibited by maltose, turanose and glucose. The enzyme released glucose residues from [3H]Glc2Man9(GlcNAc)2 but did not release glucose from [3H]Glc3Man9(GlcNAc)2. Reductive SDS-PAGE revealed two closely associated major bands showing mol. wt. of 66 and 64 ksSa. These bands also showed PAS-positive staining. Gel filtration revealed a molecular size of 290 kDa for purified enzyme

  6. Identification of Mannheimia haemolytica Adhesins Involved in Binding to Bovine Bronchial Epithelial Cells▿

    OpenAIRE

    Kisiela, Dagmara I.; Czuprynski, Charles J.

    2008-01-01

    Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by...

  7. Citrus-derived oil inhibits Staphylococcus aureus growth and alters its interactions with bovine mammary cells.

    Science.gov (United States)

    Federman, C; Joo, J; Almario, J A; Salaheen, S; Biswas, D

    2016-05-01

    This experiment examined the effects of cold-pressed, terpeneless citrus-derived oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of bovine mammary cells (MAC-T). To determine minimum inhibitory concentration, we used the broth dilution method, using CDO concentrations range from 0.0125 to 0.4% with 2-fold dilutions. Growth inhibition was examined by adding 0.00, 0.05, 0.025, 0.0125, and 0.00625% CDO to 10(5) cfu/mL S. aureus in nutrient broth and enumerating colonies after serial dilution. In a 96-well plate, S. aureus (10(7) cfu/mL) was allowed to form a biofilm, treated with 0, 0.025, 0.5, or 1% CDO, and then was measured using a spectrophotometer. Cytotoxic effect on immortalized MAC-T cells was also examined at various concentrations of CDO using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We observed that the minimum inhibitory concentration of CDO to inhibit the growth of S. aureus in vitro was 0.025% CDO. A time kill curve for CDO action on S. aureus over 4h was generated. The CDO completely eliminated S. aureus after 3h of incubation at a concentration of 0.25%, or after 2h of incubation at concentrations of 0.05%. It was also observed that CDO had no effect on preformed biofilms except at a concentration of 0.05%, in which a significant reduction in the measured absorbance was noted. In addition, the association and invasion of S. aureus to MAC-T cells were significantly inhibited after 1h of treatment with CDO. Citrus-derived oil was also able to increase cellular proliferation of MAC-T cells at concentrations up 0.05% and had no effect at a concentration of 0.1% after 1 h. Our data suggests that CDO should be considered for further research as a preventive and therapeutic against bovine mastitis. PMID:26947297

  8. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Arieh Riskin

    2015-10-01

    Full Text Available Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC in culture. Methods: Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC. To study GLUT1 targeting and recycling in living mouse MEC (MMEC in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM, or exposed to secretion medium (SM, containing prolactin. Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

  9. Alternative splicing regulated by butyrate in bovine epithelial cells.

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    Sitao Wu

    Full Text Available As a signaling molecule and an inhibitor of histone deacetylases (HDACs, butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT and control (CT groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001 at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor and Exon#11 (Acceptor in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC

  10. Canine Mammary Cancer Stem Cells are Radio- and Chemo-Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Lisa Y., E-mail: lisa.pang@ed.ac.uk; Cervantes-Arias, Alejandro; Else, Rod W.; Argyle, David J. [Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG (United Kingdom)

    2011-03-30

    Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology.

  11. Canine Mammary Cancer Stem Cells are Radio- and Chemo-Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype

    International Nuclear Information System (INIS)

    Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology

  12. INSULIN ANALOGUES: ANALYSIS OF PROLIFERATIVE POTENCY AND CHARACTERIZATION OF RECEPTORS AND SIGNALLING PATHWAYS ACTIVATED IN HUMAN MAMMARY EPITHELIAL CELLS

    OpenAIRE

    Shukla, Ashish

    2009-01-01

    Insulin analogues have been developed with the aim to provide better glycaemic control to diabetic patients. They are generated by modifying the insulin backbone which, however, may alter relevant biochemical characteristics such as the affinity to insulin receptor and type I insulin-like growth factor receptor (IGF-IR), and the insulin receptor dissociation rate. As a result insulin analogues may exhibit stronger mitogenic potency than regular insulin. Normal mammary epithelial cells show hi...

  13. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells.

    OpenAIRE

    Band, V; De Caprio, J A; Delmolino, L; Kulesa, V; Sager, R

    1991-01-01

    We have shown previously that introduction of the human papillomavirus type 16 (HPV16) or HPV18 genome into human mammary epithelial cells induces their immortalization. These immortalized cells have reduced growth factor requirements. We report here that transfection with a single HPV16 gene E6 is sufficient to immortalize these cells and reduce their growth factor requirements. The RB protein is normal in these cells, but the p53 protein is sharply reduced, as shown by immunoprecipitation w...

  14. Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.

    OpenAIRE

    Yoshimura, M.; Oka, T

    1990-01-01

    To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kilobases (kb) of the 5'-flanking sequence and 1.6 kb of the 3'-flanking sequence of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyl-transferase (CAT) gene. The chimeric gene was transfected by the calcium phosphate-precipitation procedure into primary mouse mammary epithelial cells prepared from pregnant mice. The transfection procedure had ...

  15. Low-dose BPA exposure alters the mesenchymal and epithelial transcriptomes of the mouse fetal mammary gland.

    Directory of Open Access Journals (Sweden)

    Perinaaz R Wadia

    Full Text Available Exposure of rodent fetuses to low doses of the endocrine disruptor bisphenol A (BPA causes subtle morphological changes in the prenatal mammary gland and results in pre-cancerous and cancerous lesions during adulthood. To examine whether the BPA-induced morphological alterations of the fetal mouse mammary glands are a associated with changes in mRNA expression reflecting estrogenic actions and/or b dependent on the estrogen receptor α (ERα, we compared the transcriptomal effects of BPA and the steroidal estrogen ethinylestradiol (EE2 on fetal mammary tissues of wild type and ERα knock-out mice. Mammary glands from fetuses of dams exposed to vehicle, 250 ng BPA/kg BW/d or 10 ng EE2/kg BW/d from embryonic day (E 8 were harvested at E19. Transcriptomal analyses on the ductal epithelium and periductal stroma revealed altered expression of genes involved in the focal adhesion and adipogenesis pathways in the BPA-exposed stroma while genes regulating the apoptosis pathway changed their expression in the BPA-exposed epithelium. These changes in gene expression correlated with previously reported histological changes in matrix organization, adipogenesis, and lumen formation resulting in enhanced maturation of the fat-pad and delayed lumen formation in the epithelium of BPA-exposed fetal mammary glands. Overall similarities in the transcriptomal effects of BPA and EE2 were more pronounced in the epithelium, than in the stroma. In addition, the effects of BPA and EE2 on the expression of various genes involved in mammary stromal-epithelial interactions were suppressed in the absence of ERα. These observations support a model whereby BPA and EE2 act directly on the stroma, which expresses ERα, ERβ and GPR30 in fetal mammary glands, and that the stroma, in turn, affects gene expression in the epithelium, where ERα and ERβ are below the level of detection at this stage of development.

  16. Characterization of MCF mammary epithelial cells overexpressing the Arylhydrocarbon receptor (AhR

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    Matsumura Fumio

    2009-07-01

    Full Text Available Abstract Background Recent reports indicate the existence of breast cancer cells expressing very high levels of the Arylhydrocarbon receptor (AhR, a ubiquitous intracellular receptor best known for mediating toxic action of dioxin and related pollutants. Positive correlation between the degree of AhR overexpression and states of increasing transformation of mammary epithelial cells appears to occur in the absence of any exogenous AhR ligands. These observations have raised many questions such as why and how AhR is overexpressed in breast cancer and its physiological roles in the progression to advanced carcinogenic transformation. To address those questions, we hypothesized that AhR overexpression occurs in cells experiencing deficiencies in normally required estrogen receptor (ER signaling, and the basic role of AhR in such cases is to guide the affected cells to develop orchestrated cellular changes aimed at substituting the normal functions of ER. At the same time, the AhR serves as the mediator of the cell survival program in the absence of ER signaling. Methods We subjected two lines of Michigan Cancer Foundation (MCF mammary epithelial cells to 3 different types ER interacting agents for a number of passages and followed the changes in the expression of AhR mRNA. The resulting sublines were analyzed for phenotypical changes and unique molecular characteristics. Results MCF10AT1 cells continuously exposed to 17-beta-estradiol (E2 developed sub-lines that show AhR overexpression with the characteristic phenotype of increased proliferation, and distinct resistance to apoptosis. When these chemically selected cell lines were treated with a specific AhR antagonist, 3-methoxy-4-nitroflavone (MNF, both of the above abnormal cellular characteristics disappeared, indicating the pivotal role of AhR in expressing those cellular phenotypes. The most prominent molecular characteristics of these AhR overexpressing MCF cells were found to be

  17. PTX3 is up-regulated in epithelial mammary cells during S. aureus intramammary infection in goat

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    Joel Fernando Soares Filipe

    2015-07-01

    mammary gland epithelial cells, and in macrophages. During S. aureus infection PTX3 was up-regulated by epithelial cells. Macrophages and mammary secretum didn’t show PTX3 modulation, but PMNs recruited during infection were variably intensely positive.PTX3 mRNA expression was low in healthy organs and tissues of goats as has been reported indeed the molecules commonly induced after pro-inflammatory stimulation. As expected, PTX3 was constitutively expressed in bone marrow, rich in PMNs and monocytes, in aorta covered by endothelium and in the skin.PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, demonstrating that it represents a first line of immune defense in goat udder. No modulation was observed in macrophages, in the secretum and in the ductal epithelial cells.Further experiments are needed to elucidate the role of PTX3 in the pathogenesis of S. aureus infection.

  18. Human breast cancer cells are redirected to mammary epithelial cells upon interaction with the regenerating mammary gland microenvironment in-vivo.

    Directory of Open Access Journals (Sweden)

    Karen M Bussard

    Full Text Available Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display 'normal' behavior when placed into 'normal' ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for 'normal' gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.

  19. Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Larivee Siobhan

    2006-05-01

    Full Text Available Abstract Introduction Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. Methods HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT. The immortalized cells were passaged in vitro and studied by a combination of G- banding and Spectral Karyotyping (SKY. H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. Results Immortal HMEC alone were not tumorigenic in γ-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. Conclusion Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.

  20. Alpha-ketoglutarate enhances milk protein synthesis by porcine mammary epithelial cells.

    Science.gov (United States)

    Jiang, Qian; He, Liuqin; Hou, Yongqing; Chen, Jiashun; Duan, Yehui; Deng, Dun; Wu, Guoyao; Yin, Yulong; Yao, Kang

    2016-09-01

    Alpha-ketoglutarate (AKG), a key intermediate in the Krebs cycle, has been reported to promote protein synthesis through activating mechanistic targeting of rapamycin (mTOR) in enterocytes. The study tested the hypothesis that AKG may enhance growth and milk protein synthesis in porcine mammary epithelial cells (PMECs). PMECs were cultured for 96 h in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing prolactin (2 µg/ml) and AKG (0 or 1.5 mM). At the end of 96-h culture, the abundance of apoptosis-related proteins (caspase-3, caspase-9), milk-specific proteins (α-lactalbumin and β-casein), mTOR signaling proteins (mTOR, p-mTOR, PERK, p-PERK, eIF2a, P70S6K and p-P70S6K), and endoplasmic reticulum stress (ERS)-associated proteins (BiP and CHOP) in PMEC were determined. Addition of AKG dose-dependently enhanced cell viability in the absence or presence of prolactin, with optimal concentrations of AKG being at 1.0 and 1.5 mM, respectively. In the presence of prolactin, addition of 1.5 mM AKG: (1) decreased (P milk protein and lactose, while relieving (P milk protein production by modulating mTOR and ERS signaling pathways in PMECs. PMID:27188418

  1. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    International Nuclear Information System (INIS)

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

  2. Ethanolamine requirement of mammary epithelial cells is due to reduced activity of base exchange enzyme

    International Nuclear Information System (INIS)

    Epithelial cells and some of their transformed derivatives require ethanolamine (Etn) to proliferate normally in defined culture medium. The amount of cellular phosphatidylethanolamine (PtdEtn) is considerably reduced when these cells are cultured without Etn. Using Etn-responsive and -nonresponsive rat mammary carcinoma cell lines, the biochemical mechanism of Etn-responsiveness of investigated. The incorporation of [3H]serine into phosphatidylserine (PtdSer) and PtdEtn in Etn-responsive cells was 60 and 37%, respectively, of those in Etn-nonresponsive cells. There was no significant difference between the two cell types in the activities of enzymes involved in PtdEtn synthesis via CDP-Etn. The activity of PtdSer decarboxylase was also very similar in these two cell types. When these cells were cultured in the presence of [32P]PtdEtn, the rate of accumulation of [32P]-labeled PtdSer from the radioactive PtdEtn was considerably reduced in Etn-responsive cells as compared to Etn-nonresponsive cells. Whereas there was no significant difference in the accumulation of the labeled PtdSer from [32P]phosphatidylcholine. These results demonstrate that the Etn-responsiveness is due to a limited ability to synthesize PtdSer resulting from a limited base exchange activity utilizing PtdEtn

  3. The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction.

    OpenAIRE

    Meier, V S; Groner, B.

    1994-01-01

    Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -...

  4. Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus, Escherichia coli and the regulating effect of taurine on these bioprocesses.

    Science.gov (United States)

    Zheng, Liuhai; Xu, Yuanyuan; Lu, Jinye; Liu, Ming; Bin Dai; Miao, Jinfeng; Yin, Yulong

    2016-07-01

    Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are important pathogens causing subclinical and clinical bovine mastitis, respectively. Taurine, an organic acid found in animal tissues, has been used for the treatment of various superficial infections and chronic inflammations. We challenged a bovine mammary epithelial cell (MEC) line (MAC-T) or a mouse mammary epithelial cell line (EpH4-Ev) with either E. coli or S. aureus and compared the responses of MECs to these 2 pathogens. We also examined the regulatory effects of taurine on these responses. Receptor analyses showed that both TLR2 and TLR4 are upregulated upon exposure to either E. coli or S. aureus. Taurine pre-treatment dampened upregulation to some extent. E. coli and S. aureus stimulated comparable levels of ROS, which could be inhibited by taurine pre-treatment. E. coli infection elicited a dramatic change in iNOS expression. Taurine significantly decreased iNOS expression in the S. aureus challenged group. Protein microarray demonstrated that 32/40 and 8/40 inflammatory molecules/mediators were increased after E. coli or S. aureus challenge, respectively. The fold changes of most molecules were higher in the E. coli infection group than that in the S. aureus infection group. Taurine negatively regulated the inflammatory profile in both bacterial infections. Pro-inflammatory cytokines (such as TNF-α) connected with TLR activation were down-regulated by taurine pre-treatment. The influence of TAK-242 and OxPAPC on cytokine/molecule expression profiles to E. coli challenge are different than to S. aureus. Some important factors (MyD88, TNF-α, IL-1β, iNOS and IL-6) mediated by TLR activation were suppressed either in protein microarray or special assay (PCR/kits) or both. TAK-242 restrained ROS production and NAGase activity similar to the effect of taurine in E. coli challenge groups. The detection of 3 indices (T-AOC, SOD and MDA) reflecting oxidative stress in vivo, showed that

  5. TGF-β1 promotes bovine mammary fibroblast proliferation through the ERK 1/2 signalling pathway.

    Science.gov (United States)

    Gao, Yuanyuan; Wang, Yuping; Li, Yingying; Xia, Xiaojing; Zhao, Shuang; Che, Yanyi; Sun, Yingying; Lei, Liancheng

    2016-07-01

    The abnormal proliferation of bovine mammary fibroblasts (BMFBs) impairs mammary gland development and lactation. Severe manifestations develop into breast fibrosis, leading to the culling of cows and causing serious losses to the dairy industry. Transforming growth factor β1 (TGF-β1) is an important modulator of cell proliferation and extracellular matrix formation; however, limited information is available on BMFBs. In this study, a convenient and stable culture method for BMFBs was established. Treatment with 5 ng/mL of TGF-β1 significantly promoted the proliferation of BMFBs and accelerated the cell cycle. TGF-β1 stimulation for up to 12 h significantly increased the relative ERK1/2 mRNA expression and enhanced the protein expression of p-ERK1/2 and cyclin D1. Conversely, the ERK1/2 inhibitor PD98059 blocked these TGF-β1 effects. Further exploration using a mouse model showed that TGF-β1 significantly increased the proportion of fibroblasts and accelerating the cell transition from the G1 to G2/M phases. In addition, TGF-β1 enhanced the expression of fibrosis markers, α-SMA and I Collagen, which could be blocked efficiently by the PD98059 in mouse mammary gland. Finally, immunofluorescence analysis confirmed that TGF-β1 promoted fibroblast proliferation in healthy dairy cows after normal long-term dietary corn straw roughage supplementation. It is suggested that the diet may promote mammary fibroblast proliferation by raising the level of TGF-β1. Our study provides new insights into how nutrition causes undesirable changes in mammary gland structure. PMID:27063575

  6. Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line

    International Nuclear Information System (INIS)

    Transforming growth factor beta (TGF-β) plays an essential role in a wide array of cellular processes. The most well studied TGF-β response in normal epithelial cells is growth inhibition. In some cell types, TGF-β induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-β, rendering NMuMG cells a good model system for studying these TGF-β effects. A National Institutes of Aging mouse 15,000 cDNA microarray was used to profile the gene expression of NMuMG cells treated with TGF-β1 for 1, 6, or 24 hours. Data analyses were performed using GenePixPro and GeneSpring software. Selected microarray results were verified by northern analyses. Of the 15,000 genes examined by microarray, 939 were upregulated or downregulated by TGF-β. This represents approximately 10% of the genes examined, minus redundancy. Seven genes previously not known to be regulated by TGF-β at the transcriptional level (Akt and RhoB) or not at all (IQGAP1, mCalpain, actinin α3, Ikki, PP2A-PR53), were identified and their regulation by TGF-β verified by northern blotting. Cell cycle pathway examination demonstrated downregulation of cyclin D2, c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. Examination of cell adhesion-related genes revealed upregulation of c-Jun, α-actinin, actin, myosin light chain, p120cas catenin (Catns), α-integrin, integrin β5, fibronectin, IQGAP1, and mCalpain. Using a cDNA microarray to examine TGF-β-regulated gene expression in NMuMG cells, we have shown regulation of multiple genes that play important roles in cell cycle control and EMT. In addition, we have identified several novel TGF-β-regulated genes that may mediate previously unknown TGF-β functions

  7. MicroRNA-206 is differentially expressed in Brca1-deficient mice and regulates epithelial and stromal cell compartments of the mouse mammary gland.

    Science.gov (United States)

    Wronski, A; Sandhu, G K; Milevskiy, M J G; Brewster, B L; Bridge, J A; Shewan, A M; Edwards, S L; French, J D; Brown, M A

    2016-01-01

    Depletion of Brca1 leads to defects in mouse mammary gland development and mammary tumors in humans and mice. To explore the role of microRNAs (miRNAs) in this process, we examined the mammary glands of MMTV-Cre Brca1(Co/Co) mice for differential miRNA expression using a candidate approach. Several miRNAs were differentially expressed in mammary tissue at day 1 of lactation and in mammary epithelial cell lines in which Brca1 messenger RNA (mRNA) levels have been reduced. Functional studies revealed that several of these miRNAs regulate mammary epithelial cell function in vitro, including miR-206. Creation and analysis of MMTV-miR-206 transgenic mice showed no effect on lactational mammary development and no tumors, but indicates a role in mammary tissue remodeling in mature mice, potentially involving Igf-1 and Sfrp1. These results indicate the potential of miRNAs to mediate the consequences of Brca1 loss and suggest a novel function for miR-206. PMID:27043663

  8. Thyroid hormone responsive (THRSP) promotes the synthesis of medium-chain fatty acids in goat mammary epithelial cells.

    Science.gov (United States)

    Yao, D W; Luo, J; He, Q Y; Wu, M; Shi, H B; Wang, H; Wang, M; Xu, H F; Loor, J J

    2016-04-01

    In nonruminants, thyroid hormone responsive (THRSP) is a crucial protein for cellular de novo lipogenesis. However, the role of THRSP in regulating the synthesis of milk fatty acid composition in goat mammary gland remains unknown. In the present study, we compared gene expression of THRSP among different goat tissues. Results revealed that THRSP had the highest expression in subcutaneous fat, and expression was higher during lactation compared with the dry period. Overexpression of THRSP upregulated the expression of fatty acid synthase (FASN), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acyltransferase 2 (DGAT2), and glycerol-3-phosphate acyltransferase (GPAM) in goat mammary epithelial cells. In contrast, overexpression of THRSP led to downregulation of thrombospondin receptor (CD36) and had no effect on the expression of acetyl-coenzyme A carboxylase α (ACACA) and sterol regulatory element binding transcription factor1 (SREBF1). In addition, overexpressing THRSP in vitro resulted in a significant increase in triacylglycerol (TAG) concentration and the concentrations of C12:0 and C14:0. Taken together, these results highlight an important role of THRSP in regulating lipogenesis in goat mammary epithelial cells. PMID:26851858

  9. Transgenic Mammary Epithelial Osteopontin (Spp1) Expression Induces Proliferation and Alveologenesis

    OpenAIRE

    Hubbard, Neil E; Chen, Qian J.; Sickafoose, Laura K.; Wood, Meghan B.; Gregg, Jeffrey P; Abrahamsson, Ninnie M.; Engelberg, Jesse A; Walls, Judith E; Borowsky, Alexander D.

    2013-01-01

    Osteopontin (OPN) Spp1 is involved in differentiation of the mammary gland. We engineered mice to overexpress OPN in mammary epithelium and describe an altered mammary phenotype. Three transgenic (Tg) founder lines FVB/N Tg(MMTV-Opn)(1-3BOR) were propagated after FVB/NJ pronuclear injections. Mammary glands from Tg-OPN mice compared to littermate controls showed, at 4 weeks of age, exaggerated terminal end buds; at 8 and 12 weeks, more numerous and complex ducts with increased luminal protein...

  10. The Effect of Storage Temperature and Time on the Concentrations of Bovine Serum Amyloid A and Its Mammary Associated Isoform

    Directory of Open Access Journals (Sweden)

    Csilla Tóthová

    2012-01-01

    Full Text Available The objective of this study was to evaluate the effect of storage under various conditions on the concentrations of major bovine acute phase protein—serum amyloid A, and its mammary isoform. Blood samples were taken from seven clinically healthy calves, and milk samples from six clinically healthy dairy cows. The harvested blood serum and the milk samples were fractioned into aliquots. One aliquot was analyzed on the day of collection without storage. The second aliquots were stored at 4°C for 1 day, the remaining aliquots were kept frozen at −18°C for 2, 7, 14, and 21 days, and then analyzed. Blood serum was analyzed for serum amyloid A (SAA. The concentrations of mammary isoform of SAA (M-SAA were measured in milk samples. Over time, the concentrations of SAA in serum showed a tendency of significant decrease during storage at −18°C (P<0.01. Similarly, the values of M-SAA decreased significantly in samples maintained at freezer temperatures (P<0.001. In the refrigerated samples, we found non-significantly lower values of SAA, as well as M-SAA. Presented results indicate that the storage temperature and duration may markedly affect the concentrations of bovine SAA and M-SAA.

  11. Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth.

    Science.gov (United States)

    Plichta, Kristin A; Mathers, Jessica L; Gestl, Shelley A; Glick, Adam B; Gunther, Edward J

    2012-11-15

    The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RAS(G12V) together with a nuclear fluorescent reporter. Monitoring of H-RAS(G12V)-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RAS(G12V) drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia. PMID:23010075

  12. Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep.

    Science.gov (United States)

    Addis, Maria Filippa; Pisanu, Salvatore; Marogna, Gavino; Cubeddu, Tiziana; Pagnozzi, Daniela; Cacciotto, Carla; Campesi, Franca; Schianchi, Giuseppe; Rocca, Stefano; Uzzau, Sergio

    2013-09-01

    Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals. PMID:23774600

  13. Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

    OpenAIRE

    Hynes, N E; Taverna, D.; Harwerth, I M; Ciardiello, F; Salomon, D S; Yamamoto, T.; Groner, B

    1990-01-01

    The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of t...

  14. Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity

    OpenAIRE

    1991-01-01

    Functional differentiation in mammary epithelia requires specific hormones and local environmental signals. The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production. To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cell...

  15. Microphysiometry Studies of Rapid Binding of Insulin-Like Growth Factor I by Parental and Transfected Mammary Epithelial Cell Lines

    OpenAIRE

    Robinson, Rose Marie

    1998-01-01

    Breast cancer is a leading cause of cancer death of women in the U.S. today. Members of the family of insulin-like growth factors (IGFs) are proposed to play a major role in the development and subsequent uncontrolled proliferation of breast cancer cells. Insulin-like growth factor-I (IGF-I) is known to be a potent mitogen for mammary epithelial cells. IGF-I acts by binding to cell surface receptors, thereby stimulating a cascade of events leading to cell division. In the interest of inte...

  16. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.

    OpenAIRE

    M. L. Li; Aggeler, J; Farson, D A; Hatier, C; Hassell, J; Bissell, M.J.

    1987-01-01

    When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on released type I collagen gels show greatly enhanced mRNA levels and secretion rates of beta-casein and of some other milk proteins. We show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows greater than 90% of cells to produce hi...

  17. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M.J.

    1997-08-11

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

  18. Effects of interferon-tau and steroids on cytochrome P450 activity in bovine endometrial epithelial cells

    Science.gov (United States)

    The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-t (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were tr...

  19. Aurora A Kinase Regulates Mammary Epithelial Cell Fate by Determining Mitotic Spindle Orientation in a Notch-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Joseph L. Regan

    2013-07-01

    Full Text Available Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland.

  20. The effects of Brazilian propolis on etiological agents of mastitis and the viability of bovine mammary gland explants.

    Science.gov (United States)

    Fiordalisi, Samira A L; Honorato, Luciana A; Loiko, Márcia R; Avancini, César A M; Veleirinho, Maria B R; Machado Filho, Luiz C P; Kuhnen, Shirley

    2016-03-01

    The objective of this study was to evaluate in vitro the antimicrobial activity of Brazilian propolis from Urupema, São Joaquim, and Agua Doce (Santa Catarina State) and green propolis from Minas Gerais State, and the effects of propolis on bovine mammary gland explant viability. The propolis samples differed in flavonoid content and antioxidant activity. Green propolis showed the highest content of flavonoids, followed by the sample from São Joaquim. The propolis from Urupema showed the lowest flavonoid content along with the lowest antioxidant activity. The total phenolics were similar across all studied samples. Despite phytochemical differences, the propolis samples from Minas Gerais, São Joaquim, and Urupema presented the same level of antimicrobial activity against Staphylococcus aureus strains. The reduction in S. aureus growth was, on average, 1.5 and 4 log10 times at 200 and 500 μg/mL, respectively. At concentrations of 1,000 μg/mL, all propolis reduced bacterial growth to zero. On the other hand, when the propolis were tested against strains of Escherichia coli, the samples presented weak antimicrobial activity. Mammary explants were maintained in culture for 96h without a loss in viability, demonstrating the applicability of the model in evaluating the toxicity of propolis. The origin and chemical composition of the propolis had an effect on mammary explant viability. We encountered inhibitory concentrations of 272.4, 171.8, 63.85, and 13.26 μg/mL for the propolis from Água Doce, Urupema, São Joaquim, and Mina Gerais, respectively. A clear association between greater antimicrobial activity and toxicity for mammary explants was observed. Of all propolis tested, the Urupema sample was noteworthy, as it showed antimicrobial activity at less toxic concentrations than the other samples, reducing bacterial growth to an average of 9.3 × 10(2) cfu/mL after 6h of contact using 200 μg/mL of extract. The results demonstrate the potential for Brazilian

  1. Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs).

    Science.gov (United States)

    Shi, Huaiping; Shi, Hengbo; Luo, Jun; Wang, Wei; Haile, Abiel B; Xu, Huifen; Li, Jun

    2014-07-01

    Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and 'S' sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n=60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (Pmodel cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. PMID:24889218

  2. The 18-kDa translocator protein (TSPO disrupts mammary epithelial morphogenesis and promotes breast cancer cell migration.

    Directory of Open Access Journals (Sweden)

    Xiaoting Wu

    Full Text Available Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO, are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864 and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer.

  3. The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation

    Directory of Open Access Journals (Sweden)

    Danila Coradini

    2014-02-01

    Full Text Available During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG and Polycomb (PcG group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.

  4. The Fibronectin-Binding Proteins of Staphylococcus aureus May Promote Mammary Gland Colonization in a Lactating Mouse Model of Mastitis

    OpenAIRE

    Brouillette, Eric; Talbot, Brian G.; Malouin, François

    2003-01-01

    The fibronectin-binding proteins (FnBPs) of Staphylococcus aureus are believed to be implicated in the pathogen's adherence to and colonization of bovine mammary glands, thus leading to infectious mastitis. In vitro studies have shown that FnBPs help the adhesion of the pathogen to bovine mammary epithelial cells. However, the importance of FnBPs for the infection of mammary glands has never been directly established in vivo. In this study with a mouse model of mastitis, the presence of FnBPs...

  5. Age and the means of bypassing stasis are determinants of the intrinsic subtypes of immortalized human mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Jonathan K Lee

    2015-03-01

    Full Text Available Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis are independent determinants of subtype in immortalized human

  6. Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

    Science.gov (United States)

    Lee, Jonathan K; Garbe, James C; Vrba, Lukas; Miyano, Masaru; Futscher, Bernard W; Stampfer, Martha R; LaBarge, Mark A

    2015-01-01

    Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells

  7. The Integrin-Mediated ILK-Parvin-αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells.

    Science.gov (United States)

    Rooney, Nicholas; Wang, Pengbo; Brennan, Keith; Gilmore, Andrew P; Streuli, Charles H

    2016-11-01

    Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1-integrin (β1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of β1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in β1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc. PMID:27019299

  8. Measuring bovine mammary gland blood flow using a transit time ultrasonic flow probe.

    Science.gov (United States)

    Gorewit, R C; Aromando, M C; Bristol, D G

    1989-07-01

    Lactating cattle were used to validate a transit time ultrasonic blood flow metering system for measuring mammary gland arterial blood flow. Blood flow probes were surgically placed around the right external pudic artery. An electromagnetic flow probe was implanted in tandem with the ultrasonic probe in two cows for comparative measurements. The absolute accuracy of the implanted flow probes was assessed in vivo by mechanical means on anesthetized cows after 2 to 3 wk of implantation. The zero offset of the ultrasonic probes ranged from -12 to 8 ml/min. When the ultrasonic probe was properly implanted, the slopes of the calibration curves were linear and ranged from .92 to .95, tracking absolute flow to within 8%. The transit time instrument's performance was examined under a variety of physiological conditions. These included milking and hormone injections. The transit time ultrasonic flow meter accurately measured physiological changes in mammary arterial blood flow in chronically prepared conscious cattle. Blood flow increased 29% during milking. Epinephrine decreased mammary blood flow by 90 to 95%. Oxytocin doses increased mammary blood flow by 15 to 24%. PMID:2674232

  9. Proteomics data in support of the quantification of the changes of bovine milk proteins during mammary gland involution.

    Science.gov (United States)

    Boggs, Irina; Hine, Brad; Smolenksi, Grant; Hettinga, Kasper; Zhang, Lina; Wheeler, Thomas T

    2016-09-01

    Here we provide data from three proteomics techniques; two-dimensional electrophoresis (2-DE) followed by identification of selected spots using PSD MALDI-TOF MS/MS, one-dimensional gel electrophoresis followed by LC-MS/MS analysis of gel slices (GeLC) and dimethyl isotopic labelling of tryptic peptides followed by Orbitrap MS/MS (DML), to quantify the changes in the repertoire of bovine milk proteins that occurs after drying off. We analysed skim milk and whey sampled at day 0 and either day 3 or day 8 after drying off. These analyses identified 45 spots by MALDI-TOF, 51 proteins by GeLC and 161 proteins by DML, for which the detailed data work-up is presented as three Excel files. The data supplied in this article supports the accompanying publication "Changes in the repertoire of bovine milk proteins during mammary involution" (Boggs et al., 2015) [1]. Data are available via ProteomeXchange with identifiers ProteomeXchange: PXD003110 and ProteomeXchange: PXD003011. PMID:27274532

  10. Ozone-induced augmentation of eicosanoid metabolism in epithelial cells from bovine trachea

    International Nuclear Information System (INIS)

    Epithelial injury and inflammation have been implicated in ozone-induced airway hyperresponsiveness. Because ozone is relatively insoluble and highly reactive, toxicologic effects of this compound may be limited to the plasma membranes of airway epithelium. We hypothesize that oxidant damage to epithelium may result in elaboration of various eicosanoids, which are known to alter airway smooth muscle responsiveness and epithelial cell functions (including ion transport). To examine eicosanoid metabolism after exposure to 0.1 to 10.0 ppm ozone, epithelial cells derived from bovine trachea were isolated and grown to confluency. Bovine tracheal cells in culture expressed differentiated features characteristic of epithelial cells, including a plasma membrane with a specialized polar morphology, an extensive network of filaments that were connected through intercellular junctional complexes, and keratin-containing monofilaments as determined by indirect immunofluorescent localization. Monolayers were alternately exposed to ozone and culture medium for 2 h in a specially designed in vitro chamber using a rotating inclined platform. Eicosanoid products were measured by the release of [3H]-labeled products from cells incubated with [3H]-arachidonic acid for 24 h before exposure and by the release of immunoreactive products into the cell supernatant. Both methods revealed ozone-induced increases in cyclooxygenase and lipoxygenase product formation with significant increases in prostaglandins E2, F2 alpha, 6-keto F1 alpha, and leukotriene B4. Release rates of immunoreactive products were dose-dependent, and ozone concentrations as low as 0.1 ppm produced an increase in prostaglandin F2 alpha. These findings are consistent with the hypothesis that ozone can augment eicosanoid metabolism in airway epithelial cells

  11. Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells.

    Science.gov (United States)

    Allegra, Maryline; Zaragkoulias, Andreas; Vorgia, Elena; Ioannou, Marina; Litos, Gabriele; Beug, Hartmut; Mavrothalassitis, George

    2012-10-01

    Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-β (TGF-β) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-β-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-β-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-β signaling through Smads was mostly unaffected, and ERF suppressed the TGF-β-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-β-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-β-signaling pathways. PMID:22875994

  12. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  13. Prolactin and glucocorticoid signaling induces lactation-specific tight junctions concurrent with β-casein expression in mammary epithelial cells.

    Science.gov (United States)

    Kobayashi, Ken; Tsugami, Yusaku; Matsunaga, Kota; Oyama, Shoko; Kuki, Chinatsu; Kumura, Haruto

    2016-08-01

    Alveolar mammary epithelial cells (MECs) in mammary glands are highly specialized cells that produce milk for suckling infants. Alveolar MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components after parturition. In the formation process of less permeable TJs, MECs show a selective downregulation of Cldn4 and a localization change of Cldn3. To investigate what induces less permeable TJs through these compositional changes in Cldns, we focused on two lactogenesis-related hormones: prolactin (Prl) and glucocorticoids. Prl caused a downregulation of Cldn3 and Cldn4 with the formation of leaky TJs in MECs in vitro. Prl-treated MECs also showed low β-casein expression with the activation of STAT5 signaling. By contrast, dexamethasone (Dex), a glucocorticoid analogue, upregulated Cldn3 and Cldn4, concurrent with the formation of less permeable TJs and the activation of glucocorticoid signaling without the expression of β-casein. Cotreatment with Prl and Dex induced the selective downregulation of Cldn4 and the concentration of Cldn3 in the region of TJs concurrent with less permeable TJ formation and high β-casein expression. The inhibition of Prl secretion by bromocriptine in lactating mice induced the upregulation of Cldn3 and Cldn4 concurrent with the downregulation of milk production. These results indicate that the coactivation of Prl and glucocorticoid signaling induces lactation-specific less permeable TJs concurrent with lactogenesis. PMID:27130254

  14. Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

    International Nuclear Information System (INIS)

    Protein secretion by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a limited understanding of the cellular processes that regulate protein secretion. In this study, we utilize human epithelial mammary cell (HMEC) lines that were engineered to express different levels of HER1, HER2 and HER3. Using an ELISA microarray platform, we evaluate the effects of epidermal growth factor family receptor (HER) expression on protein secretion in the HMEC lines upon initiation of HER1 receptor activation. The secreted proteins include three HER1 ligands, interleukins 1α and 18, RANTES, vascular-endothelial and platelet-derived growth factors, matrix metalloproteases 1, 2 and 9, and the extracellular portion of the HER1 and HER2 proteins. In addition, we investigate whether MAPK/Erk and PI3K/Akt signaling regulate protein secretion in these cell lines and if so, whether the involvement of HER2 or HER3 receptor alters their response to MAPK/Erk and PI3K/Akt signal pathway inhibition in terms of protein secretion. Differential expression of HER2 and HER3 receptors alters the secretion of a variety of growth factors, cytokines, and proteases. Some alterations in protein secretion are still observed when MAPK/Erk or PI3K/Akt signaling is inhibited. This study suggests that HER overexpression orchestrates broad changes in the tumor microenvironment by altering the secretion of a diverse variety of biologically active proteins

  15. Adenosine-5'-triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells.

    Science.gov (United States)

    Craddick, Michael; Patel, Rakhi; Lower, Amanda; Highlander, Sarah; Ackermann, Mark; McClenahan, David

    2012-09-15

    Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1. PMID:22771196

  16. Inhibitory effect of fluvoxamine on β-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

    Science.gov (United States)

    Chiba, Takeshi; Maeda, Tomoji; Kimura, Soichiro; Morimoto, Yasunori; Sanbe, Atsushi; Ueda, Hideo; Kudo, Kenzo

    2015-11-01

    Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on β-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 μM fluvoxamine for 72 h significantly decreased protein levels of β-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 μM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in β-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 μM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of β-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses β-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells. PMID:26415980

  17. Old and new stories: revelations from functional analysis of the bovine mammary transcriptome during the lactation cycle.

    Directory of Open Access Journals (Sweden)

    Massimo Bionaz

    Full Text Available The cow mammary transcriptome was explored at -30, -15, 1, 15, 30, 60, 120, 240, and 300 d relative to parturition. A total of 6,382 differentially expressed genes (DEG at a false discovery rate ≤ 0.001 were found throughout lactation. The greatest number of DEG (>3,500 DEG was observed at 60 and 120 d vs. -30 d with the largest change between consecutive time points observed at -15 vs. 1 d and 120 vs. 240 d. Functional analysis of microarray data was performed using the Dynamic Impact Approach (DIA. The DIA analysis of KEGG pathways uncovered as the most impacted and induced 'Galactose metabolism', 'Glycosylphosphatidylinositol (GPI-anchor biosynthesis', and 'PPAR signaling'; whereas, 'Antigen processing and presentation' was among the most inhibited. The integrated interpretation of the results suggested an overall increase in metabolism during lactation, particularly synthesis of carbohydrates and lipid. A marked degree of utilization of amino acids as energy source, an increase of protein export, and a decrease of the protein synthesis machinery as well cell cycle also were suggested by the DIA analysis. The DIA analysis of Gene Ontology and other databases uncovered an induction of Golgi apparatus and angiogenesis, and the inhibition of both immune cell activity/migration and chromosome modifications during lactation. All of the highly-impacted and activated functions during lactation were evidently activated at the onset of lactation and inhibited when milk production declined. The overall analysis indicated that the bovine mammary gland relies heavily on a coordinated transcriptional regulation to begin and end lactation. The functional analysis using DIA underscored the importance of genes associated with lactose synthesis, lipid metabolism, protein synthesis, Golgi, transport, cell cycle/death, epigenetic regulation, angiogenesis, and immune function during lactation.

  18. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    C Lin

    Full Text Available Our previous studies showed that bovine respiratory syncytial virus (BRSV followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2 epithelial cells with H. somni concentrated culture supernatant (CCS stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2 and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

  19. Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

    2009-06-03

    PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

  20. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    OpenAIRE

    Shaokun Zhang; Zaoxia Liu; Guanfang Su; Hong Wu

    2016-01-01

    The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human prima...

  1. Host microenvironment in breast cancer development: Epithelial-cell–stromal-cell interactions and steroid hormone action in normal and cancerous mammary gland

    International Nuclear Information System (INIS)

    Mammary epithelial cells comprise the functional component of the normal gland and are the major target for carcinogenesis in mammary cancer. However, the stromal compartment of the normal gland and of tumors plays an important role in directing proliferative and functional changes in the epithelium. In vivo and in vitro studies of the murine mammary gland have provided insights into novel stroma-dependent mechanisms by which estrogen and progesterone action in the epithelium can be modulated by hepatocyte growth factor (HGF) and the extracellular matrix proteins, collagen type I, fibronectin and laminin. In vitro and in vivo studies of estrogen receptor positive, estrogen-responsive human breast cancer cells have also demonstrated that estrogen responsiveness of tumor cells can also be modulated by extracellular matrix proteins, collagen type I and laminin

  2. Comparison of methods to differentiate staphylococcus and micrococcus species isolated from bovine mammary glands

    OpenAIRE

    Pengov A.; Jurčevič A.

    2003-01-01

    The hemolytic pattern of colonies, the slide coagulase (clumping factor) test, the tube coagulase test and the thermonuclease test were studied for their capability to differentiate Staphylococcus aureus from other Staphylococcus and Micrococcus species. A total 93 strains of Staphylococcus aureus isolated from bovine udder glands were tested. The hemolytic pattern of colonies and the slide coagulase test were found to be less reliable than other tests. No important differences could be found...

  3. Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Talhouk Rabih S

    2010-11-01

    Full Text Available Abstract Background IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO, an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. Methods SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET for different time periods and analyzed for induction of IL-6 secretion and NO production by ELISA and Griess reaction, respectively. The expression of IL-6 and induced NO synthase (iNOS was assayed by real time PCR and/or western immunoblots, and the activation of NFκB was assayed by immunobinding assay. To investigate the role of mammary cell microenvironment (cell-substratum or interaction of mammary epithelial cell types; critical to mammary development, function, and disease in modulation of the inflammatory response, SCp2 cells were cultured with or without extracellular matrix (EHS or in coculture with their myoepithelial counterpart (SCg6, and assayed for ET-induced IL-6 and NO. Results Endotoxin induced NFκB activation at 1 h after ET application. IL-6 secretion and NO production were induced, but with unexpected delay in expression of mRNA for iNOS compared to IL-6. NFκB/p65 activation was transient but NFκB/p50 activation persisted longer. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in IL-6 and iNOS regulation via NFκB. SCp2 cells in coculture with SCg6 but not in presence of EHS dramatically induced IL-6 secretion even in the absence of ET. ET-induced NO production was blunted in SCp2/SCg6 cocultures compared to that in SCp2 alone. Conclusions The differential regulation of IL-6 and iNOS together with the

  4. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line.

    OpenAIRE

    Doppler, W; Groner, B; Ball, R K

    1989-01-01

    We have detected hormone response elements in the promoter region of the rat beta-casein gene that confer the synergistic action of prolactin and glucocorticoid hormones upon transcription of chimeric gene constructs. A 2800-base-pair (bp) rat beta-casein gene fragment containing 2300 bp of 5' flanking sequence was placed in front of a chloramphenicol acetyltransferase (CAT) reporter gene and stably transfected into the mouse mammary epithelial cell line HC11. Addition of prolactin or dexamet...

  5. Comparison of methods to differentiate staphylococcus and micrococcus species isolated from bovine mammary glands

    Directory of Open Access Journals (Sweden)

    Pengov A.

    2003-01-01

    Full Text Available The hemolytic pattern of colonies, the slide coagulase (clumping factor test, the tube coagulase test and the thermonuclease test were studied for their capability to differentiate Staphylococcus aureus from other Staphylococcus and Micrococcus species. A total 93 strains of Staphylococcus aureus isolated from bovine udder glands were tested. The hemolytic pattern of colonies and the slide coagulase test were found to be less reliable than other tests. No important differences could be found between the tube coagulase test and the thermonuclease test. The API-Staph test was used as a reference.

  6. Milk fatty acid composition and mammary lipogenic genes expression in bovine cloned and control cattle

    OpenAIRE

    Bernard, Laurence; Richard, Christophe; Gelin, Valerie; Leroux, Christine; Heyman, Yvan

    2015-01-01

    In order to understand the effect of nuclear transfer technology on the physiology of lactation and milk fatty acid composition in dairy cattle (Holstein breed), the present study compares the milk yield and composition from bovine somatic cell cloned (n=5) and control animals (n=5) at 180 days in milk (DIM) maintained together under the same conditions. All cows were offered the same total mixed ration ad libitum. At 180 DIM, the cloned had a higher body weight (BW; P<0.01) compared with ...

  7. The effect of oxythioquinox exposure on normal human mammary epithelial cell gene expression: A microarray analysis study

    Directory of Open Access Journals (Sweden)

    Weston Ainsley

    2004-09-01

    Full Text Available Abstract Background Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures. Methods Gene expression was studied in primary normal human mammary epithelial cells (NHMECs derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix™ and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI. DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR of ± 0.6 and p value ≤ 0.05 in three of four cell strains analyzed. Results RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated. Cluster analysis examined the effects of OTQ on the cells with specific p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased at one or more time point by at least a 0.6 signal log ratio (SLR. The intermediate variant

  8. Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells - Implications for milk cholesterol secretion

    DEFF Research Database (Denmark)

    Mani, O; Körner, M; Ontsouka, C E;

    2011-01-01

    test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at...

  9. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shaokun Zhang

    2016-01-01

    Full Text Available The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.

  10. Chitosan modification of adenovirus to modify transfection efficiency in bovine corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    I-Jong Wang

    Full Text Available BACKGROUND: The purpose of this study is to modulate the transfection efficiency of adenovirus (Ad on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS. METHODOLOGY/PRINCIPAL FINDINGS: Modified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS. Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from -24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization. CONCLUSIONS/SIGNIFICANCE: Chitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications.

  11. Fetal irradiation of rats induces persistent translocations in mammary epithelial cells similar to the level after adult irradiation, but not in hematolymphoid cells.

    Science.gov (United States)

    Nakano, Mimako; Nishimura, Mayumi; Hamasaki, Kanya; Mishima, Shuji; Yoshida, Mitsuaki; Nakata, Akifumi; Shimada, Yoshiya; Noda, Asao; Nakamura, Nori; Kodama, Yoshiaki

    2014-02-01

    In both humans and mice, fetal exposure to radiation fails to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level response to radiation exposure is counterintuitive in view of the generally accepted belief that a fetus is sensitive to radiation. To determine if this is a general phenomenon, both mammary epithelial cells and spleen cells were studied in rats. Fetuses of 17.5 days postcoitus were irradiated with 2 Gy of gamma rays, and mammary tissues were removed 6-45 weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH method. The results showed that translocation frequencies were not only elevated in rats irradiated as fetuses, but were also almost as high as those in rats that were irradiated as adults (12 weeks old, pregnant mothers or young virgins) and examined 6-45 weeks later. There was no evidence of higher sensitivity in fetal cells with respect to the induction of translocations. In contrast, translocation frequencies in spleen cells were not elevated in adult rats irradiated as fetuses but were increased after irradiation of adults as previously seen in mouse spleen cells and human T lymphocytes. In the case of irradiation of adult rats, the induced translocation frequencies were similar between spleen cells and mammary epithelial cells. If we take translocation frequency as a surrogate marker of potential carcinogenic effect of radiation, the current results suggest that fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but this does not contribute to the increased risk of cancer since it has been reported that irradiation of fetal rats of the SD strain does not increase the risk of mammary cancers. Possible reasons for this discrepancy are discussed. PMID:24512615

  12. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Directory of Open Access Journals (Sweden)

    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  13. EGF-Receptor-Mediated Mammary Epithelial Cell Migration is Driven by Sustained ERK Signaling from Autocrine Stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Joslin, Elizabeth J.; Opresko, Lee; Wells, Alan; Wiley, H. S.; Lauffenburger, Douglas A.

    2007-10-15

    Aberrant expression of epidermal growth factor (EGF) receptor family ligands, as well as the receptors themselves, has been implicated in various types of cancers. EGF family ligands are synthesized as membrane-anchored proteins requiring proteolytic release to form the mature soluble factor. Despite the pathophysiological importance of autocrine systems, how the rate of protease-mediated ligand release quantitatively influences receptor-mediated signaling and consequent cell behavior is poorly understood. Therefore, we explored the relationship between autocrine EGF release rates and receptor-mediated ERK activation and migration in human mammary epithelial cells. A quantitative spectrum of EGF release rates was achieved using a set of chimeric transmembrane EGF ligand precursors modulated by the addition of the metalloprotease inhibitor batimastat. We found that ERK activation increased with increasing ligand release rates despite concomitant EGF receptor downregulation. Cell migration speed depended linearly on the steady-state phospho-ERK level obtained from either autocrine or exogenous ligand, but was much greater at any given phospho-ERK level for autocrine compared to exogenous stimulation. In contrast, cell proliferation rates were relatively constant across the various treatment conditions. Thus, in these cells, ERK-mediated migration stimulated by EGF receptor signaling is most sensitively regulated by autocrine ligand control mechanisms.

  14. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    When primary mouse mammary epithelial cells are cultured on plastic, they rapidly lose their ability to synthesize and secrete most milk proteins even in the presence of lactogenic hormones, whereas cells cultured on release type I collagen gels show greatly enhanced mRNA levels and secretion rates of β-casein and of some other milk proteins. The authors show here that culture on a reconstituted basement membrane from Engelbreth-Holm-Swarm tumor (EHS) allows > 90% of cells to produce high levels of β-casein. By comparison, 30-40% of cells on released type 1 gels and only 2-10% of cells on plastic express β-casein after 6 days in culture. Because only 40% of cells from late pregnant gland produced β-casein before culture, the EHS matrix can both induce and maintain an increased level of casein gene expression. Individual basal lamina components were also evaluated. Type IV collagen and fibronectin had little effect on morphology and β-casein mRNA levels. In contrast, both laminin and heparan sulfate proteoglycan increased β-casein mRNA levels. Profound morphological differences were evident between cells cultured on plastic and on EHS matrix, the latter cells forming ducts, ductules, and lumina and resembling secretory alveoli. These results emphasize the vital role of the extracellular matrix in receiving and integrating structural and functional signals that can direct specific gene expression in differentiated tissues

  15. Short communication: Altered expression of specificity protein 1 impairs milk fat synthesis in goat mammary epithelial cells.

    Science.gov (United States)

    Zhu, J J; Luo, J; Xu, H F; Wang, H; Loor, J J

    2016-06-01

    Specificity protein 1 (encoded by SP1) is a novel transcription factor important for the regulation of lipid metabolism and the normal function of various hormones in model organisms. Its potential role, if any, on ruminant milk fat is unknown. Despite the lower expression of the lipolysis-related gene ATGL (by 44 and 37% respectively), both the adenoviral overexpression and the silencing of SP1 [via short interfering (si)RNA] markedly reduced cellular triacylglycerol (TAG) content (by 28 and 25%, respectively), at least in part by decreasing the expression of DGAT1 (-36% in adenovirus treatment) and DGAT2 (-81 and -87%, respectively) that are involved in TAG synthesis. Consistent with the markedly lower expression of genes related to lipid droplet formation and secretion (TIP47 by 19 and 32%, and ADFP by 25 and 25%, respectively), cellular lipid droplet content was also decreased sharply, by 9 and 8.5%, respectively, after adenoviral overexpression of SP1 or its silencing via siRNA. Overall, the results underscored a potentially important role of SP1 in maintaining milk-fat droplet synthesis in goat mammary epithelial cells. PMID:26995134

  16. Visible micro-Raman spectroscopy of single human mammary epithelial cells exposed to x-ray radiation

    Science.gov (United States)

    Delfino, Ines; Perna, Giuseppe; Lasalvia, Maria; Capozzi, Vito; Manti, Lorenzo; Camerlingo, Carlo; Lepore, Maria

    2015-03-01

    A micro-Raman spectroscopy investigation has been performed in vitro on single human mammary epithelial cells after irradiation by graded x-ray doses. The analysis by principal component analysis (PCA) and interval-PCA (i-PCA) methods has allowed us to point out the small differences in the Raman spectra induced by irradiation. This experimental approach has enabled us to delineate radiation-induced changes in protein, nucleic acid, lipid, and carbohydrate content. In particular, the dose dependence of PCA and i-PCA components has been analyzed. Our results have confirmed that micro-Raman spectroscopy coupled to properly chosen data analysis methods is a very sensitive technique to detect early molecular changes at the single-cell level following exposure to ionizing radiation. This would help in developing innovative approaches to monitor radiation cancer radiotherapy outcome so as to reduce the overall radiation dose and minimize damage to the surrounding healthy cells, both aspects being of great importance in the field of radiation therapy.

  17. Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-ling; LIU Feng-jun; ZHANG Yong

    2009-01-01

    [Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%; MGE: 33.33% vs. 6.67%). Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%), confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.

  18. The Balance of Cell Surface and Soluble Type III TGF-β Receptor Regulates BMP Signaling in Normal and Cancerous Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Catherine E. Gatza

    2014-06-01

    Full Text Available Bone morphogenetic proteins (BMPs are members of the TGF-β superfamily that are over-expressed in breast cancer, with context dependent effects on breast cancer pathogenesis. The type III TGF-β receptor (TβRIII mediates BMP signaling. While TβRIII expression is lost during breast cancer progression, the role of TβRIII in regulating BMP signaling in normal mammary epithelium and breast cancer cells has not been examined. Restoring TβRIII expression in a 4T1 murine syngeneic model of breast cancer suppressed Smad1/5/8 phosphorylation and inhibited the expression of the BMP transcriptional targets, Id1 and Smad6, in vivo. Similarly, restoring TβRIII expression in human breast cancer cell lines or treatment with sTβRIII inhibited BMP-induced Smad1/5/8 phosphorylation and BMP-stimulated migration and invasion. In normal mammary epithelial cells, shRNA-mediated silencing of TβRIII, TβRIII over-expression, or treatment with sTβRIII inhibited BMP-mediated phosphorylation of Smad1/5/8 and BMP induced migration. Inhibition of TβRIII shedding through treatment with TAPI-2 or expression of a non-shedding TβRIII mutant rescued TβRIII mediated inhibition of BMP induced Smad1/5/8 phosphorylation and BMP induced migration and/or invasion in both in normal mammary epithelial cells and breast cancer cells. Conversely, expression of a TβRIII mutant, which exhibited increased shedding, significantly reduced BMP-mediated Smad1/5/8 phosphorylation, migration, and invasion. These data demonstrate that TβRIII regulates BMP-mediated signaling and biological effects, primarily through the ligand sequestration effects of sTβRIII in normal and cancerous mammary epithelial cells and suggest that the ratio of membrane bound versus sTβRIII plays an important role in mediating these effects.

  19. Effects of Cooling and Supplemental Bovine Somatotropin on Milk Production relating to Body Glucose Metabolism and Utilization of Glucose by the Mammary Gland in Crossbred Holstein Cattle

    Directory of Open Access Journals (Sweden)

    Siravit Sitprija

    2010-01-01

    Full Text Available Problem statement: The low milk yield and shorter persistency of lactation of dairy cattle is the major problem for the dairy practices in the tropics. High environmental temperatures and rapid decline of plasma growth hormone level can influence milk production. Regulation of the milk yield of animals is mainly based on the mechanisms governing the quantity of glucose extracted by the mammary gland for lactose biosynthetic pathways. The mechanism(s underlying the effects of cooling and supplemental bovine somatotropin on milk production relating to body glucose metabolism and intracellular metabolism of glucose in the mammary gland of crossbred Holstein cattle in the tropics have not been investigated to date. Approach: Ten crossbred 87.5% Holstein cows were divided into two groups of five animals each. Animals were housed in Normal Shade barn (NS as non-cooled cows and cows in the second group were housed in barn which was equipped with a two Misty-Fan cooling system (MF as cooled cows. Supplementation of recombinant bovine Somatotropin (rbST (POSILAC, 500 mg per cow were performed in both groups to study body glucose metabolism and the utilization of glucose in the mammary gland using a continuous infusion of [3-3H] glucose and [U- 14C] glucose as markers in early, mid and late stages of lactation. Results: Milk yield significantly increased in both groups during supplemental rbST with a high level of mammary blood flow. Body glucose turnover rates were not significant different between cooled and non-cooled cows whether supplemental rbST or not. The glucose taken up by the mammary gland of both non-cooled and cooled cows increased flux through the lactose synthesis and the pentose cycle pathway with significant increases in NADPH formation for fatty acid synthesis during rbST supplementation. The utilization of glucose carbon incorporation into milk appeared to increase in milk lactose and milk triacylglycerol but not for

  20. Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

    Science.gov (United States)

    Shandilya, Umesh K; Sharma, Ankita; Sodhi, Monika; Kapila, Neha; Kishore, Amit; Mohanty, Ashok; Kataria, Ranjit; Malakar, Dhruva; Mukesh, Manishi

    2016-02-01

    Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues

  1. Insulin receptors in the mammary gland

    International Nuclear Information System (INIS)

    Insulin binding studies were conducted using mammary membrane preparations to further the authors understanding of insulin's role in regulating mammary metabolism, particularly ruminant mammary metabolism. Specific objectives were to: (1) characterize insulin binding to bovine mammary microsomes and determine if the specificity and kinetics of binding indicate the presence of insulin receptors in bovine mammary gland; (2) examine and compare insulin binding by liver and mammary microsomes of the pig and dairy cow; (3) examine insulin binding to bovine milk fat globule membranes (MFGM) and evaluate this model's usefulness in assessing insulin receptor regulation in the mammary gland of the cow; (4) examine the effect of dietary fat in insulin binding by rat mammary and liver microsomes. The specificity and kinetics of 125I-insulin binding of bovine mammary microsomes indicated the presence of insulin receptors in bovine mammary gland. Bovine liver and mammary microsomes specifically bound less 125I-insulin than did the corresponding porcine microsomes, and mammary microsomes, regardless of species, specifically bound less 125I-insulin than did liver microsomes. These differences in binding suggest differences in insulin responsiveness between pigs and cattle, as well as between the liver and mammary glands

  2. Mammary gland stem cells

    DEFF Research Database (Denmark)

    Fridriksdottir, Agla J R; Petersen, Ole W; Rønnov-Jessen, Lone

    2011-01-01

    understood. The mouse is a widely used model of mammary gland development, both directly by studying the mouse mammary epithelial cells themselves and indirectly, by studying development, morphogenesis, differentiation and carcinogenesis of xenotransplanted human breast epithelium in vivo. While in early...... develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology....

  3. Paracrine signaling through the epithelial estrogen receptor α is required for proliferation and morphogenesis in the mammary gland

    OpenAIRE

    Mallepell, Sonia; Krust, Andrée; Chambon, Pierre; Brisken, Cathrin

    2006-01-01

    Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor α (ERα) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERα mutant strain that retains some of the protein with transactivation activity. Here, we use an ERα−/− mouse strain in which no ERα transcript can be detected to analyze mammary gland development in the complete absence of ERα signaling. The ER...

  4. Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

    Directory of Open Access Journals (Sweden)

    Tentler John J

    2011-08-01

    regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.

  5. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    Science.gov (United States)

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  6. Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells.

    Science.gov (United States)

    Severson, Paul L; Vrba, Lukas; Stampfer, Martha R; Futscher, Bernard W

    2014-12-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. PMID:25435355

  7. Transgenic mice exhibiting inducible and spontaneous Cre activities driven by a bovine keratin 5 promoter that can be used for the conditional analysis of basal epithelial cells in multiple organs

    Directory of Open Access Journals (Sweden)

    Liang Chih-Chia

    2009-01-01

    Full Text Available Abstract Background Cre/loxP-mediated genetic modification is the most widely used conditional genetic approach used in the mouse. Engineered Cre and the mutated ligand-binding domain of estrogen receptor fusion recombinase (CreERT allow temporal control of Cre activity. Results In this study, we have generated two distinct transgenic mouse lines expressing CreERT, which show 4-hydroxytamoxifen (4-OHT-inducible and spontaneous (4-OHT-independent Cre activities, referred to Tg(BK5-CreERTI and Tg(BK5-CreERTS, respectively. The transgenic construct is driven by the bovine Keratin 5 promoter, which is active in the basal epithelial lineage of stratified and pseudo-stratified epithelium across multiple organs. Despite the difference in 4-OHT dependency, the Tg(BK5-CreERTI and Tg(BK5-CreERTS mouse lines shared similar Cre-mediated recombination among various organs, except for unique mammary epithelial Cre activity in Tg(BK5-CreERTS females. Conclusion These two new transgenic mouse lines for the analysis of basal epithelial function and for the genetic modification have been created allowing the identification of these cell lineages and analysis of their differentiation during embryogenesis, during perinatal development and in adult mice.

  8. Bovine lactoferrin regulates cell survival, apoptosis and inflammation in intestinal epithelial cells and preterm pig intestine

    DEFF Research Database (Denmark)

    Nguyen, Duc Ninh; Jiang, Pingping; Stensballe, Allan; Bendixen, Emøke; Sangild, Per T.; Chatterton, Dereck E. W.

    2016-01-01

    Bovine lactoferrin (bLF) may modulate neonatal intestinal inflammation. Previous studies in intestinal epithelial cells (IECs) indicated that moderate bLF doses enhance proliferation whereas high doses trigger inflammation. To further elucidate cellular mechanisms, we profiled the porcine IEC...... proteome after stimulation with bLF at 0, 0.1, 1 and 10g/L by LC-MS-based proteomics. Key pathways were analyzed in the intestine of formula-fed preterm pigs with and without supplementation of 10g/L bLF. Levels of 123 IEC proteins were altered by bLF. Low bLF doses (0.1-1g/L) up-regulated 11 proteins...... acid metabolism, and altered three proteins enhancing the hypoxia inducible factor-1 (HIF-1) pathway. In the preterm pig intestine, bLF at 10g/L decreased villus height/crypt depth ratio and up-regulated the Bax/Bcl-2 ratio and HIF-1α, indicating elevated intestinal apoptosis and inflammation. In...

  9. Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry.

    Science.gov (United States)

    Peña, L; Gama, A; Goldschmidt, M H; Abadie, J; Benazzi, C; Castagnaro, M; Díez, L; Gärtner, F; Hellmén, E; Kiupel, M; Millán, Y; Miller, M A; Nguyen, F; Poli, A; Sarli, G; Zappulli, V; de las Mulas, J Martín

    2014-01-01

    Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms. PMID:24227007

  10. Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

    OpenAIRE

    Earp H Shelton; Strunk Karen E; Hunter Debra M; Sandahl Melissa; Cook Rebecca S

    2010-01-01

    Abstract Background Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in mo...

  11. ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1

    Energy Technology Data Exchange (ETDEWEB)

    Horn, Galit; Gaziel, Avital [Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel); The Alec and Myra Marmot Hybridoma Unit, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel); Wreschner, Daniel H. [Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel); Biomodifying LLC, San Diego, CA 92122 (United States); Smorodinsky, Nechama I., E-mail: nechama@post.tau.ac.il [Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel); The Alec and Myra Marmot Hybridoma Unit, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel); Ehrlich, Marcelo [Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978 (Israel)

    2009-05-01

    Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

  12. ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1

    International Nuclear Information System (INIS)

    Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

  13. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    Science.gov (United States)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  14. Insulin-like growth factor-I and insulin-like growth factor binding proteins in the bovine mammary gland: Receptors, endogenous secretion, and appearance in milk

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, P.G.

    1988-01-01

    This is the first study to characterize both insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) in bovine milk, to characterize the IGF-I receptor in the dry and lactating mammary gland, and to report de novo synthesis and secretion of IGF-I and IGFBP from normal mammary tissue. Immunoreactive IGF-I was principally associated with 45 kDa IGFBP in milk. Multiparous cows had a higher IGF-I concentration of 307 ng/ml than primiparous cows at 147 ng/ml. IGF-I concentration on day 56 of lactation was 34 ng/ml for combined parity groups. At parturition, IGF-I mass in blood and milk pools was 1.4 and 1.2 mg, respectively. Binding of {sup 125}I-IGF-I was specific for IGF-I with anIC{sub 50} of 2.2 ng which was a 10- and 1273-fold greater affinity than IGF-II and insulin, respectively. Association constants, as determined by Scatchard analysis, were similar for both pregnant and lactating cows at 3.5 and 4.0 L/nM, respectively. In addition, estimated mean receptor concentration was 0.25 and 0.23 pM/mg protein for pregnant and lactating cows, respectively. In a survey of mammary microscomes prepared from 48 cows, {sup 125}I-IGF-I binding declined with progressing lactation and a similar trend was observed during pregnancy.

  15. Plasma transport and mammary uptake of trans fatty acids in dairy cows

    OpenAIRE

    Vargas Bello Pérez, Einar

    2011-01-01

    In this thesis, aspects of metabolism of lipids in dairy cows were studied, particularly 18:1 trans fatty acid (tFA) concentrations in plasma and lipoprotein fractions, and transportation of FA in epithelial mammary gland cell cultures. Two in vivo studies were conducted to elucidate which lipoprotein fractions were involved in bovine plasma transport of tFA by infusing oils that induced different plasma tFA profiles. Fatty acid profiles of plasma and lipoprotein fractions [high (HDL), l...

  16. MicroRNA content in milk exosomes as a phenotypic indicator of Staphylococcus aureus infection in the bovine mammary gland

    Science.gov (United States)

    Previous gene mapping research to understand the host genetic response to mammary infection based on somatic cell score has been unsuccessful due to the poor correlation of this confounding trait with mastitis, a disease costing the dairy industry an estimated $2 billion in annual costs. Recently, ...

  17. Effects of bovine mammary gland biopsy and increased milking frequency on post-procedure udder health, histology, and milk yield.

    Science.gov (United States)

    Lima, J A M; Ruas, J R M; Vasconcelos, A C; Silper, B F; Lana, A M Q; Gheller, V A; Saturnino, H M; Reis, R B; Coelho, S G

    2016-05-01

    Sixteen cows in early lactation were randomly distributed into two groups in order to evaluate the effects of mammary biopsies and increased milking frequency on tissue characteristics, post-biopsy udder health and histology. One group was milked twice a day (2×) starting on the 2nd day after calving, until 28 days in milk (DIM). The other group was milked four times a day (4×) from two to 21 DIM, and twice a day (2×) from 22 to 28 DIM. On days 2, 7, 14, 21, and 28 postpartum, one fragment of secretory tissue was collected from one mammary quarter at a time. Collections were alternated between the four mammary quarters per collection day. A total of 80 mammary tissue samples were collected. Qualitative and quantitative analyses of the tissues were conducted by histologic examination. Animal health was assessed by observation of feed intake behavior immediately after biopsy, and weight and body condition score before and one week after biopsy. Udder health was assessed daily from calving to 60 DIM with California Mastitis Test (CMT) and by noting alterations in the milk such as blood, milk clots, blood clots, clinical signs of mastitis. Milk composition and somatic cell count (SCC) were analyzed before and after the biopsies. Milk production was evaluated before biopsy, on the day of biopsy, and after the biopsy. An average of 10 fields at 40× magnification was obtained from each sample. There were no evident changes in mammary morphology as a result of milking two or four times/day at any of the evaluated time points. Biopsy wounds healed rapidly without infection. Intramammary bleeding and CMT alterations were observed in 96% and 75% of the biopsied mammary quarters, respectively. Clinical mastitis was diagnosed in 12% of the biopsied quarters. Different milking frequencies had no effect on the frequency and duration of post-biopsy alterations. Milk production decreased after biopsies done on days 2 for 2× and 4× groups, but it returned to pre-biopsy values

  18. Interactions between exosomes from breast cancer cells and primary mammary epithelial cells leads to generation of reactive oxygen species which induce DNA damage response, stabilization of p53 and autophagy in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sujoy Dutta

    Full Text Available Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive "niches". Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7, representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs. Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.

  19. Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation

    Directory of Open Access Journals (Sweden)

    Li Zhen

    2012-12-01

    Full Text Available Abstract Background MicroRNAs (miRNAs have been implicated in the regulation of milk protein synthesis and development of the mammary gland (MG. However, the specific functions of miRNAs in these regulations are not clear. Therefore, the elucidation of miRNA expression profiles in the MG is an important step towards understanding the mechanisms of lactogenesis. Results Two miRNA libraries were constructed from MG tissues taken from a lactating and a non-lactating Holstein dairy cow, respectively, and the short RNA sequences (18–30 nt in these libraries were sequenced by Solexa sequencing method. The libraries included 885 pre-miRNAs encoding for 921 miRNAs, of which 884 miRNAs were unique sequences and 544 (61.5% were expressed in both periods. A custom-designed microarray assay was then performed to compare miRNA expression patterns in the MG of lactating and non-lactating dairy cows. A total of 56 miRNAs in the lactating MG showed significant differences in expression compared to non-lactating MG (P Conclusion Our study provides a broad view of the bovine MG miRNA expression profile characteristics. Eight hundred and eighty-four miRNAs were identified in bovine MG. Differences in types and expression levels of miRNAs were observed between lactating and non-lactating bovine MG. Systematic predictions aided in the identification of lactation-related miRNAs, providing insight into the types of miRNAs and their possible mechanisms in regulating lactation.

  20. Effect of short-chain fatty acids on triacylglycerol accumulation, lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells.

    Science.gov (United States)

    Sun, Yuting; Luo, Jun; Zhu, Jiangjiang; Shi, Hengbo; Li, Jun; Qiu, Siyuan; Wang, Ping; Loor, Juan J

    2016-02-01

    Short-chain fatty acids (SCFAs) are the major energy sources for ruminants and are known to regulate various physiological functions in other species. However, their roles in ruminant milk fat metabolism are still unclear. In this study, goat mammary gland epithelial cells (GMECs) were treated with 3 mmol/L acetate, propionate or butyrate for 24 h to assess their effects on lipogenesis. Data revealed that the content of triacylglycerol (TAG) and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate and butyrate treatment. In contrast, the messenger RNA (mRNA) expression of FASN and LXRα was not affected by propionate, but reduced by butyrate. Acetate had no obvious effect on the content of TAG and lipid droplets but increased the mRNA expression of SCD1 and FABP3 in GMECs. Additionally, it was observed that propionate significantly increased the relative content of mono-unsaturated fatty acids (C18:1 and C16:1) at the expense of decreased saturated fatty acids (C16:0 and C18:0). Butyrate and acetate had no significant effect on fatty acid composition. Overall, the results from this work help enhance our understanding of the regulatory role of SCFAs on goat mammary cell lipid metabolism. PMID:26304676

  1. Pro-inflammatory cytokine TNF-α is a key inhibitory factor for lactose synthesis pathway in lactating mammary epithelial cells.

    Science.gov (United States)

    Kobayashi, Ken; Kuki, Chinatsu; Oyama, Shoko; Kumura, Haruto

    2016-01-15

    Lactose is a milk-specific carbohydrate synthesized by mammary epithelial cells (MECs) in mammary glands during lactation. Lactose synthesis is downregulated under conditions causing inflammation such as mastitis, in which MECs are exposed to high concentrations of inflammatory cytokines. In this study, we investigated whether inflammatory cytokines (TNF-α, IL-1β, and IL-6) directly influence the lactose synthesis pathway by using two types of murine MEC culture models: the monolayer culture of MECs to induce lactogenesis; and the three-dimensional culture of MECs surrounded by Matrigel to induce reconstitution of the alveolar structure in vitro. TNF-α caused severe down-regulation of lactose synthesis-related genes concurrently with the degradation of glucose transporter 1 (GLUT1) from the basolateral membranes in MECs. IL-1β also caused degradation of GLUT1 along with a decrease in the expression level of β-1,4-galactosylransferase 3. IL-6 caused both up-regulation and down-regulation of the expression levels of lactose synthesis-related genes in MECs. These results indicate that TNF-α, IL-1β, and IL-6 have different effects on the lactose synthesis pathway in MECs. Furthermore, TNF-α triggered activation of NFκB and inactivation of STAT5, suggesting that NFκB and STAT5 signaling pathways are involved in the multiple adverse effects of TNF-α on the lactose synthesis pathway. PMID:26518119

  2. A species-specific activation of Toll-like receptor signaling in bovine and sheep bronchial epithelial cells triggered by Mycobacterial infections.

    Science.gov (United States)

    Ma, Yan; Han, Fei; Liang, Jinping; Yang, Jiali; Shi, Juan; Xue, Jing; Yang, Li; Li, Yong; Luo, Meihui; Wang, Yujiong; Wei, Jun; Liu, Xiaoming

    2016-03-01

    Pulmonary tuberculosis caused by a Mycobacterium infection remains a major public health problem in most part of the world, in part owing to the transmission of its pathogens between hosts including human, domestic and wild animals. To date, molecular mechanisms of the pathogenesis of TB are still incompletely understood. In addition to alveolar macrophages, airway epithelial cells have also been recently recognized as main targets for Mycobacteria infections. In an effort to understand the pathogen-host interaction between Mycobacteria and airway epithelial cells in domestic animals, in present study, we investigated the Toll-like receptor (TLR) signaling in bovine and sheep airway epithelial cells in response to an infection of Mycobacterium tuberculosis avirulent H37Ra stain or Mycobacterium bovis BCG vaccine strain, using primary air-liquid interface (ALI) bronchial epithelial culture models. Our results revealed a host and pathogen species-specific TLR-mediated recognition of pathogen-associated molecular patterns (PAMPs), induction and activation of TLR signaling pathways, and substantial induction of inflammatory response in bronchial epithelial cells in response to Mycobacteria infections between these two species. Interestingly, the activation TLR signaling in bovine bronchial epithelial cells induced by Mycobacteria infection was mainly through a myeloid differentiation factor 88 (MyD88)-independent TLR signaling pathway, while both MyD88-dependent and independent TLR signaling cascades could be induced in sheep epithelial cells. Equally noteworthy, a BCG infection was able to induce both MyD88-dependent and independent signaling in sheep and bovine airway epithelial cells, but more robust inflammatory responses were induced in sheep epithelial cells relative to the bovines; whereas an H37Ra infection displayed an ability to mainly trigger a MyD88-independent TLR signaling cascade in these two host species, and induce a more extent expression of

  3. Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy.

    Directory of Open Access Journals (Sweden)

    Raghav K Chhetri

    Full Text Available The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A or pre-malignant (MCF10DCIS.com MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF. This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios [1] were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled in vitro, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.

  4. Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary gland fibroblasts.

    Science.gov (United States)

    Wu, Jianmei; Ding, Yulin; Bi, Yannan; Wang, Yi; Zhi, Yu; Wang, Jinling; Wang, Fenglong

    2016-06-01

    Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. To better understand the importance of bovine mammary fibroblasts (BMFBs) and the roles of the TLR-NF-κB and TLR-AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibosis, BMFBs cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA and protein expression of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMFBs. This information offers new potential targets for the treatment of bovine mammary fibrosis. PMID:26948281

  5. SOX4 Mediates TGF-beta-Induced Expression of Mesenchymal Markers during Mammary Cell Epithelial to Mesenchymal Transition

    NARCIS (Netherlands)

    Vervoort, Stephin J.; Lourenco, Ana Rita; van Boxtel, Ruben; Coffer, Paul J.

    2013-01-01

    The epithelial to mensenchymal transition program regulates various aspects of embryonic development and tissue homeostasis, but aberrant activation of this pathway in cancer contributes to tumor progression and metastasis. TGF-beta potently induces an epithelial to mensenchymal transition in cancer

  6. Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Victor O Okoh

    Full Text Available The purpose of this study was to investigate the effects of 17-β-estradiol (E2-induced reactive oxygen species (ROS on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2, a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes - nuclear respiratory factor-1 (NRF-1 was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor - NRF-1. In summary, our study has demonstrated that: (i 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2

  7. Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice

    OpenAIRE

    Robichaux, Jacqulyne P.; Hallett, Robin M; Fuseler, John W.; Hassell, John A.; Ramsdell, Ann F.

    2014-01-01

    Significant left-right (L-R) differences in tumor incidence and disease outcome occur for cancers of paired organs, including the breasts; however, the basis for this laterality is unknown. Here, we show that despite their morphological symmetry, left versus right mammary glands in wild type mice have baseline differences in gene expression that are L-R independently regulated during pubertal development, including genes that regulate luminal progenitor cell renewal, luminal cell differentiat...

  8. The ETS Transcription Factor ESE-1 Transforms MCF-12A Human Mammary Epithelial Cells via a Novel Cytoplasmic Mechanism

    OpenAIRE

    Prescott, Jason D.; Koto, Karen S. N.; Singh, Meenakshi; Gutierrez-Hartmann, Arthur

    2004-01-01

    Several different transcription factors, including estrogen receptor, progesterone receptor, and ETS family members, have been implicated in human breast cancer, indicating that transcription factor-induced alterations in gene expression underlie mammary cell transformation. ESE-1 is an epithelium-specific ETS transcription factor that contains two distinguishing domains, a serine- and aspartic acid-rich (SAR) domain and an AT hook domain. ESE-1 is abundantly expressed in human breast cancer ...

  9. 4-Vinylcyclohexene Diepoxide (VCD) Inhibits Mammary Epithelial Differentiation and Induces Fibroadenoma Formation in Female Sprague Dawley Rats

    OpenAIRE

    Wright, Laura E; Frye, Jennifer B.; Lukefahr, Ashley L; Marion, Samuel L.; Hoyer, Patricia B.; Besselsen, David G.; Funk, Janet L.

    2011-01-01

    4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, bo...

  10. Regulation of leptin in involution of mammary gland

    Institute of Scientific and Technical Information of China (English)

    LI Meng; LI Qingzhang

    2007-01-01

    Leptin, a protein hormone produced and secreted predominantly by white adipose tissue, has a critical role in the regulation and coordination of energy metabolism. Leptin is produced in the mammary gland by the fat tissue or by the mammary epithelium. In vitro study has shown that leptin triggers apoptosis in mammary epithelial cells. Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, STAT3 is specifically activated. Various studies show that leptin act as a paracrine and autocrin factor to influence mammary epithelial cell proliferation and differentiation. This paper reviewed the function of leptin to the involution of mammary gland.

  11. Survey of bovine mycotic mastitis in different mammary gland statuses in two north-eastern regions of Algeria.

    Science.gov (United States)

    Ksouri, Samir; Djebir, Somia; Hadef, Youcef; Benakhla, Ahmed

    2015-04-01

    The aim of this study was to evaluate the prevalence of mycotic mastitis in different mammary gland statuses. The study was conducted on 304 dairy cows from ten farms in two north-eastern regions in Algeria; Guelma and Souk Ahras with 922 and 199 samples, respectively, forming thus a total number of 1,121 milk samples. A total of 321 milk samples were collected from clinical mastitis, 544 milk samples from subclinical mastitis and 256 milk samples from healthy mammary glands. Mycological analyses revealed that 10.17% of the treated samples were positive recording 114 species of fungi including 88 yeasts and 26 moulds. The most frequent species was Candida kefyr followed by C. albicans, C. guilliermondii, C. famata, C. tropicalis, C. colliculosa, C. krusei, C. rugosa, C. glabrata, C. parapsilosis, C. inconspicua, Trichosporon sp., Rhodotorula glutinis and Saccharomyces fragilis. Mould species have also been isolated from samples of both healthy milk and clinical mastitis milk. Aspergillus amstelodami (from glaucus group), A. fumigatus and Geotrichum candidum were identified, while the other species including Penicillium sp. and Cladosporium sp. were not identified. PMID:25481847

  12. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  13. Milk synthetic response of the bovine mammary gland to an increase in the local concentration of amino acids and acetate.

    Science.gov (United States)

    Purdie, N G; Trout, D R; Poppi, D P; Cant, J P

    2008-01-01

    Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10 h with 0 or 40 g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0 g/h, 30 g/h in the profile of rumen microbes, or 30 g/h in the profile of milk proteins, in a 3 x 2 factorial arrangement with the 2 acetate treatments of 0 and 40 g/h, all in a 6 x 6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and beta-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields

  14. Evaluation of the Rapid Mastitis Test for identification of Staphylococcus aureus and Streptococcus agalactiae isolated from bovine mammary glands.

    OpenAIRE

    Watts, J L; Owens, W E

    1988-01-01

    A latex agglutination test system (Rapid Mastitis Test [RMT]; Immucell, Portland, Maine) containing reagents for the identification of Staphylococcus aureus and Streptococcus agalactiae from bovine intramammary infections was evaluated with 527 staphylococcal and 267 streptococcal isolates. The RMT Staphylococcus aureus reagent detected 94.2% of 242 Staphylococcus aureus isolates, 80% of 25 Staphylococcus intermedius isolates, and 42.8% of 21 tube coagulase-positive Staphylococcus hyicus isol...

  15. Effects of media and the presence of bovine oviduct epithelial cells during in vitro fertilization on fertilizability and developmental capacity of bovine oocytes.

    Science.gov (United States)

    Choi, Y H; Fukui, Y; Ono, H

    1991-11-01

    The effect of the presence of bovine oviduct epithelial cells (BOEC; Experiment 1) as well as the effects of media (Tyrode fertilization medium: TFM vs synthetic oviduct fluid: SOF), fertilization containers (drops in petri dish vs 96-wells), and the number of oocytes per drop and well (5 vs 10) for in vitro fertilization (Experiment 2) on the fertilizability and in vitro development of bovine oocytes were investigated. Immature oocytes with cumulus cells were cultured in TCM199 supplemented with 10% ECS and 2.5x10(6) granulosa cells for 24 hours at 39 degrees C under 5% CO(2) in air. In vitro fertilization was performed with frozen-thawed, heparin-treated spermatozoa (100 mug/ml, 15 minutes) and with BOEC (Experiment 1). In Experiment 2, in vitro fertilization was performed with two different media (TFM and SOF) and various conditions (culture dish and different number of oocytes). Cleavage, development to the blastocyst stage were evaluated on Day 2 and Day 7 after the start of culture. Effect of the presence of BOEC on fertilizability and developmental capacity (Experiment 1) was not significantly different. In Experiment 2, alterations in media, containers and number of oocytes during in vitro fertilization had no affect. The SOF medium showed results similar to those of TFM (normal fertilization rate: 63.2 vs 64%; cleavage: 69.3 vs 73.9%; development to the blastocyst stage: 14 vs 15%; and mean number of nuclei per blastocyst: 80.5 vs 86.6). The results indicate that the presence of BOEC during in vitro fertilization did not improve fertilizability, and that SOF as well as TFM medium can be utilized as a simple fertilization medium. PMID:16727055

  16. Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

    Directory of Open Access Journals (Sweden)

    Maiara G. Blagitz

    2015-01-01

    Full Text Available This study evaluated the expression of CD14, toll-like receptor (TLR 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

  17. Mediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections.

    Science.gov (United States)

    Detilleux, J; Theron, L; Duprez, J-N; Reding, E; Moula, N; Detilleux, M; Bertozzi, C; Hanzen, C; Mainil, J

    2016-08-01

    Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log2-transformed CFU that elicited one unit increase in log2-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log2-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components. PMID:26923826

  18. Molecular genetic analysis of VRK1 in mammary epithelial cells: depletion slows proliferation in vitro and tumor growth and metastasis in vivo.

    Science.gov (United States)

    Molitor, T P; Traktman, P

    2013-01-01

    The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family. VRK1, a ser/thr kinase with a nuclear localization, is the most well-studied paralog and has been described as a proproliferative protein. In lower eukaryotes, a loss of VRK1 activity is associated with severe mitotic and meiotic defects. Mice that are hypomorphic for VRK1 expression are infertile, and depletion of VRK1 in tissue culture cells can impair cell proliferation and alter several signaling pathways. VRK1 has been implicated as part of a 'gene-expression signature' whose overexpression correlates with poor clinical outcome in breast cancer patients. We present here our investigation of the role of VRK1 in the growth of normal (MCF10) and malignant (MDA-MB-231) human mammary epithelial cells, and demonstrate that shRNA-mediated depletion of VRK1 slows their proliferation significantly. Conversely, stable overexpression of a FLAG-tagged VRK1 transgene imparts a survival advantage to highly malignant MDA-MB-231 cells under conditions of nutrient and growth factor deprivation. Moreover, in a murine orthotopic xenograft model of breast cancer, we demonstrate that tumors depleted of VRK1 show a 50% reduction in size from 4-13 weeks postengraftment. The incidence and burden of distal metastases in the lungs and brain was also significantly reduced in mice engrafted with VRK1-depleted cells. These studies demonstrate that VRK1 depletion or overexpression has an impact on the proliferation and survival of cell lines derived from normal or malignant mammary tissue, and moreover show that depletion of VRK1 in MDA-MB-231 cells reduces their oncogenic and metastatic properties in vivo. PMID:23732708

  19. Homology with vesicle fusion mediator syntaxin-1a predicts determinants ofepimorphin/syntaxin-2 function in mammary epithelial morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Connie S.; Nelson, Celeste M.; Khauv, Davitte; Bennett, Simone; Radisky, Evette S.; Hirai, Yohei; Bissell, Mina J.; Radisky, Derek C.

    2009-06-03

    We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

  20. Epidermal growth factor-mediated T-cell factor/lymphoid enhancer factor transcriptional activity is essential but not sufficient for cell cycle progression in nontransformed mammary epithelial cells

    OpenAIRE

    Graham, Nicholas A.; Asthagiri, Anand R.

    2004-01-01

    Because beta-catenin target genes such as cyclin D1 are involved in cell cycle progression, we examined whether beta-catenin has a more pervasive role in normal cell proliferation, even upon stimulation by non-Wnt ligands. Here, we demonstrate that epidermal growth factor (EGF) stimulates T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcriptional activity in nontransformed mammary epithelial cells (MCF-10A) and that its transcriptional activity is essential for EGF-mediated progression ...

  1. Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

    OpenAIRE

    Stampfer, Martha R.; Garbe, James; Levine, Gerri; Lichtsteiner, Serge; Vasserot, Alain P.; Yaswen, Paul

    2001-01-01

    Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the ab...

  2. Expression of Putative Stem Cell Marker, Hepatocyte Nuclear Factor 4 Alpha, in Mammary Gland of Water Buffalo.

    Science.gov (United States)

    Choudhary, Ratan K; Choudhary, Shanti; Kaur, Harmanjot; Pathak, Devendra

    2016-01-01

    Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that ( 1 ) HNF4A identifies putative buffalo mammary stem/progenitor cells and ( 2 ) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5-10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4-4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify

  3. Effect of dietary fat on uptake of lysine, phenylalanine, leucine and methionine by bovine mammary tissue slices in vitro

    International Nuclear Information System (INIS)

    Four mature Holstein cows in late lactation were blocked in two groups based on milk production, in a 2x2 reversal with 21-day periods, and fed: (A) control diet; (B) A plus 1 kg/day tallow. Cows were fed sorghum silage ad libitum. Blood samples were collected from the jugular vein on day 15, 17, and 19 of each period. Fat did not effect DM intake or milk yield, however milk CP yield was 20% lower. Plasma lipids increased 33.6%, glucose decreased 9% and insulin/glucagon ratio decreased 21.2% in cow fed fat. After period two, cows were slaughtered and mammary tissue sampled for incubation in Krebs Ringer bicarbonate buffer containing 22 AA at arterial concentration and .225 μCi/ml of 14C-labelled L-Leu, L-Phe, L-Lys or D/L Met. Dietary fat decreased tissue AA uptake rate by 21.2%. Uptake was 4.8, 10.3, 17.8 and 2.4 x 10-3 μM/min/gm of tissue DM for Phe, Lys, Leu and Met, respectively. Results suggest that dietary fat may decrease milk protein synthesis by lowering the rate of AA uptake

  4. Downregulation of hepatocyte nuclear factor-4α and its role in regulation of gene expression by TGF-β in mammary epithelial cells

    International Nuclear Information System (INIS)

    We found that a specific isoform of hepatocyte nuclear factor 4α (HNF-4α), HNF-4α8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-β. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4α8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4α8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4α8 in TGF-β signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-β1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-β1 was attenuated by forced expression of HNF-4α8. HMGA2 has recently emerged as a transcriptional organizer of TGF-β signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4α as a new target downstream of HMGA2 and assigned a new role to HNF-4α in the TGF-β signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells

  5. Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Damian J Junk

    Full Text Available Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1 occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

  6. Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer

    Science.gov (United States)

    Jacca, Sarah; Rolih, Valeria; Quaglino, Elena; Franceschi, Valentina; Tebaldi, Giulia; Bolli, Elisabetta; Rosamilia, Alfonso; Ottonello, Simone; Cavallo, Federica; Donofrio, Gaetano

    2016-01-01

    ABSTRACT The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs. PMID:27141335

  7. Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation

    International Nuclear Information System (INIS)

    Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were

  8. Intramammary infusion of Panax ginseng extract in bovine mammary gland at cessation of milking induces changes in the expression of toll-like receptors, MyD88 and NF-kB during early involution.

    Science.gov (United States)

    Baravalle, Celina; Silvestrini, Paula; Cadoche, Mónica C; Beccaria, Camila; Andreotti, Carolina S; Renna, María S; Pereyra, Elizabeth A L; Ortega, Hugo H; Calvinho, Luis F; Dallard, Bibiana E

    2015-06-01

    The purposes of this study were to explore TLR2 and TLR4 participation and MyD88 and NF-κB activation in bovine mammary glands (BMG) treated with Panax ginseng (PG) at involution and verify the effect of PG in the cytokine expression. Quarters were infused at the end of lactation with PG solution (3 mg/ml), placebo or kept as uninoculated controls. Cows were slaughtered at 7 d after cessation of milking and mammary tissue samples were taken. A significant increase of TLR2, TLR4, MyD88, NF-κB, IL-1β, IL-6 and TGF-β1 mRNA expression was observed in PG-treated quarters. Immunostaining of TLR2 and TLR4 was significantly higher in PG mammary tissues. The percentages of immunopositive cells for NF-κB-p65 were significantly higher in PG-treated quarters. The BMG responded to PG extract components possibly by TLR2 and TLR4 signaling pathway. These results provide an insight into potential mechanisms by which PG stimulates innate immunity during BMG involution. PMID:25866011

  9. Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant.

    Science.gov (United States)

    Bonnefont, Cécile M D; Rainard, Pascal; Cunha, Patricia; Gilbert, Florence B; Toufeer, Mehdi; Aurel, Marie-Rose; Rupp, Rachel; Foucras, Gilles

    2012-04-01

    Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria. PMID:22337903

  10. WISP3 (CCN6) Regulates Milk Protein Synthesis and Cell Growth Through mTOR Signaling in Dairy Cow Mammary Epithelial Cells.

    Science.gov (United States)

    Jiang, Nan; Wang, Yu; Yu, Zhiqiang; Hu, Lijun; Liu, Chaonan; Gao, Xueli; Zheng, Shimin

    2015-08-01

    The mTOR/S6K1 signaling pathway is the primary regulator of milk protein synthesis. While mTOR is known to be regulated at the translational level by amino acids, the mechanism by which mTOR accepts the amino acid signal is not yet clear. In this study, we describe the discovery of WISP3 as a potentially novel signaling factor that connects mTOR and amino acids. Treatment of dairy cow mammary epithelial cells with amino acids (lysine or methionine) increased both cell growth and the expression of β-casein (CSN2), WISP3, mTOR, and phospho-mTOR (p-mTOR). Notably, overexpressing WISP3 in these cells also increased both cell growth and the expression of CSN2, mTOR, and p-mTOR and decreased the expression of glycogen synthase kinase 3β (GSK3β), while repressing WISP3 had the opposite effect. The increase of the expression of CSN2, mTOR, and p-mTOR mediated by amino acid could be inhibited by repressing WISP3. The increase of the expression of CSN2, mTOR, and p-mTOR mediated by WISP3 overexpression could be inhibited by overexpressing GSK3β, and vice versa. Taken together, these results reveal that through its amino acid-mediated regulation of the mTOR pathway, WISP3 is an important regulatory factor involved in the amino acid-mediated regulation of milk protein synthesis and cell growth. PMID:26061139

  11. Integrated analysis reveals that STAT3 is central to the crosstalk between HER/ErbB receptor signaling pathways in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Chunhong; Zhang, Yi; Shankaran, Harish; Resat, Haluk

    2015-01-01

    Human epidermal growth factor receptors (HER, also known as ErbB) drive cellular proliferation, pro-survival and stress responses by activating several downstream kinases, in particular ERK, p38, JNK (SAPK), the PI3K/AKT, as well as various transcriptional regulators such as STAT3. When co-expressed, first three members of HER family (HER1-3) can form homo- and hetero-dimers. Based on the considerable evidence which suggest that every receptor dimer activates intracellular signaling pathways differentially, we hypothesized that the HER dimerization pattern is a better predictor of downstream signaling than the total receptor activation levels. We validated our hypothesis using a combination of model-based analysis to quantify the HER dimerization patterns and multi-factorial experiments where HER dimerization patterns and signaling crosstalk were rationally perturbed. We have measured the activation of HER1-3 receptors and of the sentinel signaling proteins ERK, AKT, p38, JNK, STAT3 as a function of time in a panel of human mammary epithelial (HME) cells expressing different levels of HER1-3 stimulated with various ligand combinations. Our analysis using multiple ways of clustering the activation data has confirmed that the HER receptor dimer is a better predictor of the signaling through p38, ERK and AKT pathways than the total HER receptor expression and activation levels. Targeted inhibition studies to identify the causal effects allowed us to obtain a consensus regulatory interaction model, which revealed that STAT3 occupies a central role in the crosstalk between the studied pathways.

  12. S100A7 (Psoriasin), highly expressed in Ductal Carcinoma In Situ (DCIS), is regulated by IFN-gamma in mammary epithelial cells

    International Nuclear Information System (INIS)

    The aim of the present work was to explore signal transduction pathways used in the regulation of S100A7 (psoriasin). Members of the S100 gene family participate in many important cellular functions. Psoriasin, S100A8 (calgranulin A) and S100A9 (calgranulin B) are expressed in ductal carcinoma in situ (DCIS), as well as in the hyperproliferative skin disease, psoriasis. In the latter condition, a disturbance in the STAT pathway has recently been reported. This pathway is implicated in the regulation of IFN-gamma, widely recognized as a key cytokine in psoriasis. IFN-gamma also exerts anti-tumor action in a number of tumor cell types, including breast cancer. We therefore examined the effect of IFN-gamma and STAT-signaling on the psoriasin expression. We established a TAC2 mouse mammary epithelial cell line with tetracycline-inducible psoriasin expression (Tet-Off). Viability in cell culture was estimated using MTS assay. Protein and gene expression were evaluated by Western blotting and quantitative real-time PCR. Statistical analyses were assessed using a one-tailed, paired t-test. We report the downregulation of psoriasin by IFN-gamma in the MDA-MB-468 breast cancer cell line, as well as the downregulation of psoriasin induced by anoikis in cell lines derived from different epithelial tissues. In contrast, IFN-gamma had no suppressive effect on calgranulin A or calgranulin B. IFN-gamma is an important activator of the STAT1 pathway and we confirmed an active signaling pathway in the cell lines that responded to IFN-gamma treatment. In contrast, in the SUM190 breast carcinoma cell line, IFN-gamma did not suppress the expression of endogenous psoriasin. Moreover, a reduced phosphorylation of the STAT1 protein was observed. We showed that IFN-gamma treatment and the inhibition of the transcription factor NFkappaB had a synergistic effect on psoriasin levels. Finally, in TAC2 cells with tetracycline-induced psoriasin expression, we observed the increased viability of

  13. The transcription factor ATF3 acts as an oncogene in mouse mammary tumorigenesis

    Directory of Open Access Journals (Sweden)

    Thames Howard D

    2008-09-01

    Full Text Available Abstract Background Overexpression of the bZip transcription factor, ATF3, in basal epithelial cells of transgenic mice under the control of the bovine cytokeratin-5 (CK5 promoter has previously been shown to induce epidermal hyperplasia, hair follicle anomalies and neoplastic lesions of the oral mucosa including squamous cell carcinomas. CK5 is known to be expressed in myoepithelial cells of the mammary gland, suggesting the possibility that transgenic BK5.ATF3 mice may exhibit mammary gland phenotypes. Methods Mammary glands from nulliparous mice in our BK5.ATF3 colony, both non-transgenic and transgenic, were examined for anomalies by histopathology and immunohistochemistry. Nulliparous and biparous female mice were observed for possible mammary tumor development, and suspicious masses were analyzed by histopathology and immunohistochemistry. Human breast tumor samples, as well as normal breast tissue, were similarly analyzed for ATF3 expression. Results Transgenic BK5.ATF3 mice expressed nuclear ATF3 in the basal layer of the mammary ductal epithelium, and often developed squamous metaplastic lesions in one or more mammary glands by 25 weeks of age. No progression to malignancy was seen in nulliparous BK5.ATF3 or non-transgenic mice held for 16 months. However, biparous BK5.ATF3 mice developed mammary carcinomas with squamous metaplasia between 6 months and one year of age, reaching an incidence of 67%. Cytokeratin expression in the tumors was profoundly disturbed, including expression of CK5 and CK8 (characteristic of basal and luminal cells, respectively throughout the epithelial component of the tumors, CK6 (potentially a stem cell marker, CK10 (a marker of interfollicular epidermal differentiation, and mIRSa2 and mIRSa3.1 (markers of the inner root sheath of hair follicles. Immunohistochemical studies indicated that a subset of human breast tumors exhibit high levels of nuclear ATF3 expression. Conclusion Overexpression of ATF3 in CK5

  14. The transcription factor ATF3 acts as an oncogene in mouse mammary tumorigenesis

    International Nuclear Information System (INIS)

    Overexpression of the bZip transcription factor, ATF3, in basal epithelial cells of transgenic mice under the control of the bovine cytokeratin-5 (CK5) promoter has previously been shown to induce epidermal hyperplasia, hair follicle anomalies and neoplastic lesions of the oral mucosa including squamous cell carcinomas. CK5 is known to be expressed in myoepithelial cells of the mammary gland, suggesting the possibility that transgenic BK5.ATF3 mice may exhibit mammary gland phenotypes. Mammary glands from nulliparous mice in our BK5.ATF3 colony, both non-transgenic and transgenic, were examined for anomalies by histopathology and immunohistochemistry. Nulliparous and biparous female mice were observed for possible mammary tumor development, and suspicious masses were analyzed by histopathology and immunohistochemistry. Human breast tumor samples, as well as normal breast tissue, were similarly analyzed for ATF3 expression. Transgenic BK5.ATF3 mice expressed nuclear ATF3 in the basal layer of the mammary ductal epithelium, and often developed squamous metaplastic lesions in one or more mammary glands by 25 weeks of age. No progression to malignancy was seen in nulliparous BK5.ATF3 or non-transgenic mice held for 16 months. However, biparous BK5.ATF3 mice developed mammary carcinomas with squamous metaplasia between 6 months and one year of age, reaching an incidence of 67%. Cytokeratin expression in the tumors was profoundly disturbed, including expression of CK5 and CK8 (characteristic of basal and luminal cells, respectively) throughout the epithelial component of the tumors, CK6 (potentially a stem cell marker), CK10 (a marker of interfollicular epidermal differentiation), and mIRSa2 and mIRSa3.1 (markers of the inner root sheath of hair follicles). Immunohistochemical studies indicated that a subset of human breast tumors exhibit high levels of nuclear ATF3 expression. Overexpression of ATF3 in CK5-expressing cells of the murine mammary gland results in the

  15. Cytotoxicity, apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A.

    Science.gov (United States)

    Giromini, Carlotta; Rebucci, Raffaella; Fusi, Eleonora; Rossi, Luciana; Saccone, Francesca; Baldi, Antonella

    2016-06-01

    This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 μg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2'-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P < 0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 μg/mL) induced 35 % LDH release in MDCK cells (P < 0.05). A significant (P < 0.05) change in percentages of apoptotic BME-UV1 (10 ± 0.86) and MDCK (25 ± 0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P < 0.05) increased in BME-UV1 cells treated with 1.25 μg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines. Graphical abstract Study results overview. PMID:27154019

  16. 7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Pereira Paulo

    2004-06-01

    Full Text Available Abstract Background Connexin43 (Cx43 is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols affect gap junction intercellular communication (GJIC. Results Primary cultures of lens epithelial cells (LEC were incubated with 7-ketocholesterol (7-Keto, 25-hydroxycholesterol (25-OH or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. Conclusions Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication

  17. Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

    Science.gov (United States)

    Ge, Guang-Zhe; Xia, Hou-Jun; He, Bao-Li; Zhang, Hai-Lin; Liu, Wen-Jing; Shao, Ming; Wang, Chun-Yan; Xiao, Ji; Ge, Fei; Li, Fu-Bing; Li, Yi; Chen, Ceshi

    2016-02-01

    The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency. PMID:26296387

  18. Role of Oligopeptide Transporter 2 in Bovine Mammary Gland Phenylalanine Dipeptide Uptake%小肽转运载体2在奶牛乳腺小肽摄取中的作用研究

    Institute of Scientific and Technical Information of China (English)

    周苗苗; 吴跃明; 刘红云; 赵珂; 刘建新

    2011-01-01

    This experiment was conducted to study the role of oligopeptide transporter 2 in small peptides uptake and milk protein synthesis in bovine mammary gland. Different doses of Phe dipeptide (0 and 11. 7 |xg/mL)and DEPC (0, 0.01, 0.1, 0. 5 and 1 mmol/L) were added to the culture medium of bovine mammary gland tissues. After incubated in the experimental medium, mammary tissues and medium were collected and used for gene expression and milk protein determination, respectively. The results showed that l) Phe dipeptide increased oligopeptide transporter 2 and αsl -casein gene expression and milk protein quantity in the medium (P0.05) in free Phe group. These results indicate that Phe dipeptide can be used for synthesis of milk protein by bovine mammary gland while PepT2 may play an important role in small peptides uptake by bovine mammary gland. [Chinese Journal of Animal Nutrition, 2011, 23(8):1303-1308]%本试验旨在研究2型小肽转运载体(oligopeptide transporter 2,PepT 2)在奶牛乳腺组织吸收利用小肽合成乳蛋白过程中的作用.在体外培养的奶牛乳腺组织培养液中分别添加不同浓度的苯丙氨酸二肽( Phe-Phe)(0和11.7 μg/mL)和/或焦碳酸二乙酯(DEPC)(0、0.01、0.1、0.5和1.0 mmol/L)进行培养,试验结束后收集乳腺组织和培养液分别用于基因表达和乳蛋白合成的检测.结果表明,Phe-Phe促进了PepT 2和αs1-酪蛋白基因表达及乳蛋白合成(P<0.05);随DEPC添加浓度的升高,αs1-酪蛋白基因表达(P<0.01)和乳蛋白合成(P<0.05)显著降低;0.5 mmol/L DEPC显著降低了Phe-Phe组αs1-酪蛋白的基因表达(P<0.05)和乳蛋白合成(P<0.01)以及不添加小肽组乳蛋白合成(P<0.05),但不影响不添加小肽组αs1-酪蛋白基因表达(P>0.05).结果提示,奶牛乳腺能摄取Phe-Phe用于乳蛋白的合成,PepT 2可能在乳腺小肽摄取过程中发挥重要作用.

  19. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences

    International Nuclear Information System (INIS)

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland λgt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation

  20. Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8

    OpenAIRE

    Hanayama, Rikinari; Nagata, Shigekazu

    2005-01-01

    During the involution of mammary glands, epithelial cells undergo apoptosis and are cleared for the next cycle of lactation. The clearance of apoptotic epithelial cells is mediated by neighboring epithelial cells and by macrophages that migrate into the mammary glands. Here, we report that milk fat globule EGF factor 8 (MFG-E8), a secreted glycoprotein that binds to apoptotic cells by recognizing phosphatidylserine, was expressed by epithelial cells and macrophages in mammary glands and was i...

  1. Heterocellular interaction enhances recruitment of α and β-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

    International Nuclear Information System (INIS)

    Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (β-casein expression) was evaluated. Heterocellular interaction is critical for β-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (α-catenin, β-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although β-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and β-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear β-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of β-catenin in GJ complexes

  2. Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Tandon Apurva

    2011-08-01

    Full Text Available Abstract Background In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2 induces apoptosis in Human Papillomavirus (HPV positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive, as well as MDA-MB-231 (highly invasive human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs isolated from tissue biopsies of patients undergoing breast reduction surgery. Results AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. Conclusion AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated

  3. An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

    Directory of Open Access Journals (Sweden)

    Moores Sheri L

    2006-05-01

    Full Text Available Abstract Background Vav proteins are guanine nucleotide exchange factors (GEF for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity. Results We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation. Conclusion Our results indicate that increased migration of active Vav1 expressing cells is dependent on

  4. Mammary tumors

    International Nuclear Information System (INIS)

    Mammary neoplasia is one of the more common malignancies affecting domestic species. Despite their importance, they are often over- diagnosed, undertreated and subject to several misconceptions propagated by veterinarians and pet owners alike. Mammary neoplasia is the most frequent tumor type encountered in the female accounting for almost half of all malignancies reported. The canine has the highest incidence of mammary tumors of all domestic species. In the dog, about 65 percent of mammary tumors are benign mixed tumors, and 25 percent are carcinomas. The rest are adenomas, myoepitheliomas, and malignant mixed tumors. The age distribution of mammary tumors closely follows the age distribution of most tumors in the dog. Mammary tumors are rare in dogs 2 years old, but incidence begins to increase sharply at approximately 6 years of age. Median age at diagnosis is about 10 years. No breed predilection has been consistently reported

  5. Resident macrophages influence stem cell activity in the mammary gland

    OpenAIRE

    Gyorki, D.E.; Asselin-Labat, M.L.; Rooijen, van, J.; Lindeman, G J; Visvader, J E

    2009-01-01

    Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland ...

  6. In vitro expansion of the mammary stem/progenitor cell population by xanthosine treatment

    Directory of Open Access Journals (Sweden)

    Choudhary Ratan K

    2012-06-01

    Full Text Available Abstract Background Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. Results In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Conclusions Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.

  7. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    Science.gov (United States)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  8. Amino acids and mammary gland development: nutritional implications for milk production and neonatal growth

    OpenAIRE

    Rezaei, Reza; Wu, Zhenlong; Hou, Yongqing; Fuller W Bazer; Wu, Guoyao

    2016-01-01

    Milk is synthesized by mammary epithelial cells of lactating mammals. The synthetic capacity of the mammary gland depends largely on the number and efficiency of functional mammary epithelial cells. Structural development of the mammary gland occurs during fetal growth, prepubertal and post-pubertal periods, pregnancy, and lactation under the control of various hormones (particularly estrogen, growth hormone, insulin-like growth factor-I, progesterone, placental lactogen, and prolactin) in a ...

  9. Cytokeratin expression during mouse embryonic and early postnatal mammary gland development

    OpenAIRE

    Sun, Peng; Yuan, Yuanyang; Li, Aihua; Li, Boan; Dai, Xing

    2009-01-01

    Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progeni...

  10. Lgr5-Expressing Cells Are Sufficient and Necessary for Postnatal Mammary Gland Organogenesis

    OpenAIRE

    Vicki Plaks; Audrey Brenot; Devon A. Lawson; Linnemann, Jelena R.; Eline C. Van Kappel; Karren C. Wong; Frederic de Sauvage; Ophir D. Klein; Zena Werb

    2013-01-01

    Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerati...

  11. Mouse Mammary Tumor Virus Molecular Biology and Oncogenesis

    Directory of Open Access Journals (Sweden)

    Susan R. Ross

    2010-09-01

    Full Text Available Mouse mammary tumor virus (MMTV, which was discovered as a milk‑transmitted, infectious cancer-inducing agent in the 1930s, has been used since that time as an animal model for the study of human breast cancer. Like other complex retroviruses, MMTV encodes a number of accessory proteins that both facilitate infection and affect host immune response. In vivo, the virus predominantly infects lymphocytes and mammary epithelial cells. High level infection of mammary epithelial cells ensures efficient passage of virus to the next generation. It also results in mammary tumor induction, since the MMTV provirus integrates into the mammary epithelial cell genome during viral replication and activates cellular oncogene expression. Thus, mammary tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer.

  12. Salivary α-amylase exhibits antiproliferative effects in primary cell cultures of rat mammary epithelial cells and human breast cancer cells

    OpenAIRE

    Bertram Catharina; Hass Ralf; Fedrowitz Maren; Löscher Wolfgang

    2011-01-01

    Abstract Background Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth. Methods The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress ...

  13. Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1 in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT in Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Seiji Mori

    Full Text Available Epithelial-to-mesenchymal transition (EMT plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β and fibroblast growth factors (FGF secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2. We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

  14. The mammary cellular hierarchy and breast cancer

    OpenAIRE

    Oakes, Samantha R.; Gallego-Ortega, David; Ormandy, Christopher J.

    2014-01-01

    Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and ...

  15. Stem Cells and the Mammary Microenvironment

    OpenAIRE

    Booth, Brian W.; Boulanger, Corinne A.; Smith, Gilbert H.

    2008-01-01

    An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct-limited and lobule-limited outgrowths...

  16. Effects of retinal growth factor and of the increase of the number of subcultures on sulfated glycosaminoglycans of bovine lens epithelial cells

    International Nuclear Information System (INIS)

    Sulfated glycosaminoglycans of cultured bovine lens epithelial cells grown in the presence and in the absence of a retinal growth factor were investigated comparatively. The newly formed [35S] sulfate-labeled glycosaminoglycans were analysed in the extra-, peri- and intracellular compartments of early (4-5th) and late (17-18h) subcultures. The following results were obtained: (1) Cultured lens epithelial cells grown in the presence or in the absence of the growth factor synthesize chondroitin 4- and 6-sulfates and dermatan sulfate, with heparan sulfate as the main component, the pericellular compartments were particularly rich in heparan sulfate; (2) The distribution pattern of the glycosaminoglycans changes during successive subcultures; the proportion of heparan sulfate increases in the pericellular compartment, the dermatan sulfate to chondroitin sulfate ratio increases in all three compartments; (3) In contrast to the drastic decrease in the fibronectin levels in the presence of growth factor in the early subcultures, only minor differences were found between the glycosaminoglycan patterns for the treated and non-treated cells. ( orig.)

  17. Mammary development and breast cancer: the role of stem cells

    OpenAIRE

    Ercan, C.; J. van Diest, P.; Vooijs, M.

    2011-01-01

    The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often fou...

  18. Mammary leptin synthesis, milk leptin and their putative physiological roles

    OpenAIRE

    Bonnet, Muriel; Delavaud, Carole; Laud, Karine; Gourdou, Isabelle; Leroux, Christine; Djiane, Jean; Chilliard, Yves

    2002-01-01

    International audience This paper reviews data on mammary leptin and leptin receptor gene expression as well as on blood and milk leptin levels during the pregnancy-lactation cycle in humans, rodents and ruminants, with the aim of better understanding milk leptin origin and functions. The few published papers report that leptin may be produced by different cell types in the mammary tissue, and may act as a paracrine factor on mammary epithelial cell proliferation, differentiation and/or ap...

  19. PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

    Science.gov (United States)

    Boelens, Mirjam C; Nethe, Micha; Klarenbeek, Sjoerd; de Ruiter, Julian R; Schut, Eva; Bonzanni, Nicola; Zeeman, Amber L; Wientjens, Ellen; van der Burg, Eline; Wessels, Lodewyk; van Amerongen, Renée; Jonkers, Jos

    2016-08-23

    Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC. PMID:27524621

  20. Developmental biology: cell fate in the mammary gland

    Science.gov (United States)

    Most breast cancers have their origin in the luminal epithelial cells of the mammary gland. Defining how a master regulator controls the development of this cell lineage could provide important hints about why this should be. ...

  1. Short communication: Urea induces T helper 2 (Th2) type environment at transcriptional level and prostaglandin E2 secretion in bovine oviduct epithelial cells in culture.

    Science.gov (United States)

    Kowsar, R; Marey, M A; Shimizu, T; Miyamoto, A

    2016-07-01

    Excess dietary protein intake in early lactation dairy cows resulting in blood urea nitrogen of greater than 19 to 20mg/dL is associated with decreased fertility. Little is known about the local interference of urea in the normal immunological environment of the oviduct that provides optimal conditions for early reproductive events. A bovine oviduct epithelial cell (BOEC) culture was used to determine how urea influences immune environment. The BOEC monolayer was supplemented with low (20mg/dL) and high (40mg/dL) concentrations of urea together with ovarian steroids, estradiol (1ng/mL) and progesterone (1ng/mL), and LH (10ng/mL) at concentrations observed during the preovulatory period. The urea values used in this study were equivalent to 9.3 and 18.7mg/dL of blood urea nitrogen, which are typically common in lactating dairy cows with low or high protein intake, respectively. Stimulation of BOEC with 40mg/dL of urea induced gene expression of IL10 and IL4, epithelial-derived T helper type 2-driving (anti-inflammatory) cytokines as well as mPGES-1 expression and prostaglandin E2 (PGE2) secretion. However, urea concentrations of both 20 and 40mg/dL failed to alter the expression of IL1B and TNFA, Th1-driving cytokines, and the gene expression of TLR4. However, a concentration of 40mg/dL of urea stimulated α 1-acid glycoprotein expression, an acute phase protein. Data from this in vitro study suggest that urea, at least in part, contributes to influence the expression of some immune-related genes toward T helper type 2 type and prostaglandin E2 secretion, leading to disruption in local environment for fertilization and early embryonic development. PMID:27132094

  2. Nuclear proteins from lactating mammary glands bind to the promoter of a milk protein gene.

    OpenAIRE

    LUBON, H.; Hennighausen, L

    1987-01-01

    The gene for the whey acidic protein (WAP) is expressed specifically in the lactating mammary glands of rodents. We present evidence that nuclear proteins from mammary epithelial cells form a multiple nucleoprotein complex with the WAP gene promoter/upstream region. As monitored by mobility shifts, nuclear proteins from lactating mammary glands and from the mammary cell line MCF-7 form four high affinity complexes with a fragment spanning the region between nucleotides -175 and -88. Nuclear p...

  3. Functional Characterization of Stem Cell Activity in the Mouse Mammary Gland

    OpenAIRE

    Bruno, Robert D.; Smith, Gilbert H.

    2011-01-01

    Any portion of the mouse mammary gland is capable of recapitulating a clonally derived complete and functional mammary tree upon transplantation into an epithelial divested mammary fat-pad of a recipient host. As such, it is an ideal model tissue for the study somatic stem cell function. This review will outline what is known regarding the function of stem/progenitor cells in the mouse mammary gland, including how progenitor populations can be functionally defined, the evidence for and potent...

  4. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  5. Alpha basic crystallin expression in canine mammary tumors

    OpenAIRE

    Guvenc, Tolga; Gulbahar, Mustafa Yavuz; YARIM, Murat; Kabak, Yonca Betil; Karayigit, Onder; Sozmen, Mahmut

    2012-01-01

    The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c posit...

  6. In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis

    DEFF Research Database (Denmark)

    Buitenhuis, Albert Johannes; Rontved, Christine M.; Edwards, Stefan McKinnon;

    2011-01-01

    Background Bovine mastitis is one of the most costly and prevalent diseases affecting dairy cows worldwide. In order to develop new strategies to prevent Escherichia coli-induced mastitis, a detailed understanding of the molecular mechanisms underlying the host immune response to an E. coli.......i. to represent the acute phase response (APR) and chronic stage, respectively. Differentially expressed (DE) genes for each stage were analyzed and the DE genes detected at T=24h were also compared to data collected from two previous E. coli mastitis studies that were carried out on post mortem tissue......, all of the up-regulated transcripts were associated with tissue healing processes. Comparison of T=24h DE genes detected in the three E. coli mastitis studies revealed 248 were common and mainly involved immune response functions. KEGG pathway analysis indicated these genes were involved in 12...

  7. The effect of DDT and its metabolite (DDE) on prostaglandin secretion from epithelial cells and on contractions of the smooth muscle of the bovine oviduct in vitro

    International Nuclear Information System (INIS)

    The insecticide DDT and its metabolite (DDE), due to their lipolytic nature and resistance to biodegradation, are accumulated in the living tissues. In cows, DDT and DDE were found to affect prostaglandin (PG) secretion from the endometrium and contractions of the myometrium. In this study, the impact of both xenobiotics (0.1, 1, 10 or 100 ng/ml) on the function of epithelial cells and muscle strips of bovine oviducts from 1 to 5 day of the oestrous cycle was examined. Therefore the concentration of PGE2 and PGFM (a metabolite of PGF2α) in culture media, mRNA expression of genes involved in PGs synthesis in epithelial cells and the force and amplitude of strips contractions were measured after 2 and 24 or 48 h of incubation. Neither DDT nor DDE affected the viability of cells after 48 h (P > 0.05). Both DDT and DDE increased the concentrations of PGFM in culture medium and secretion of PGE2 after only 2 h of cell culture (P < 0.05). Similar effects were seen for the influence of DDE on amount of PGFM after 48 h, while DDT decreased secretion of PGE2 (P < 0.05). DDT after 2 h increased (P < 0.05) mRNA expression of PGF2α synthase (PGFS), while both xenobiotics decreased (P < 0.05) mRNA expression of cyclooxygenase-2 (COX-2) after 24 h. DTT also increased the force of isthmus contractions after 2 h, as did both xenobiotics after 48 h (P < 0.05). Moreover, after 2 and 48 h, DDE stimulated the amplitude of contractions of the isthmus as well as the ampulla, (P < 0.05). The effect of both compounds on oviduct contractions was diminished by indomethacin, which blocks PG synthesis. We conclude that oviductal secretion of prostaglandins is affected, by DDT and DDE. The influence of these xenobiotics on PGF2α and PGE2 secretion and ratio may be part of the mechanism by which both DDT and its metabolite disturb the contractions of oviductal muscle. -- Highlights: ► DDT and its metabolite – DDE are accumulated in the living tissues. ► The insecticides affected PGF2

  8. The effect of DDT and its metabolite (DDE) on prostaglandin secretion from epithelial cells and on contractions of the smooth muscle of the bovine oviduct in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wrobel, Michal H.; Mlynarczuk, Jaroslaw; Kotwica, Jan, E-mail: janko@pan.olsztyn.pl

    2012-03-01

    The insecticide DDT and its metabolite (DDE), due to their lipolytic nature and resistance to biodegradation, are accumulated in the living tissues. In cows, DDT and DDE were found to affect prostaglandin (PG) secretion from the endometrium and contractions of the myometrium. In this study, the impact of both xenobiotics (0.1, 1, 10 or 100 ng/ml) on the function of epithelial cells and muscle strips of bovine oviducts from 1 to 5 day of the oestrous cycle was examined. Therefore the concentration of PGE2 and PGFM (a metabolite of PGF2α) in culture media, mRNA expression of genes involved in PGs synthesis in epithelial cells and the force and amplitude of strips contractions were measured after 2 and 24 or 48 h of incubation. Neither DDT nor DDE affected the viability of cells after 48 h (P > 0.05). Both DDT and DDE increased the concentrations of PGFM in culture medium and secretion of PGE2 after only 2 h of cell culture (P < 0.05). Similar effects were seen for the influence of DDE on amount of PGFM after 48 h, while DDT decreased secretion of PGE2 (P < 0.05). DDT after 2 h increased (P < 0.05) mRNA expression of PGF2α synthase (PGFS), while both xenobiotics decreased (P < 0.05) mRNA expression of cyclooxygenase-2 (COX-2) after 24 h. DTT also increased the force of isthmus contractions after 2 h, as did both xenobiotics after 48 h (P < 0.05). Moreover, after 2 and 48 h, DDE stimulated the amplitude of contractions of the isthmus as well as the ampulla, (P < 0.05). The effect of both compounds on oviduct contractions was diminished by indomethacin, which blocks PG synthesis. We conclude that oviductal secretion of prostaglandins is affected, by DDT and DDE. The influence of these xenobiotics on PGF2α and PGE2 secretion and ratio may be part of the mechanism by which both DDT and its metabolite disturb the contractions of oviductal muscle. -- Highlights: ► DDT and its metabolite – DDE are accumulated in the living tissues. ► The insecticides affected PGF2

  9. Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor.

    OpenAIRE

    Ulich, T. R.; Yi, E. S.; Cardiff, R; Yin, S.; Bikhazi, N.; Biltz, R; Morris, C. F.; Pierce, G. F.

    1994-01-01

    Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF is secreted by stromal cells and affects epithelial but not mesenchymal cell proliferation. KGF injected intravenously was found to cause dramatic proliferation of mammary epithelium in the mammary glands of rats. KGF causes ductal neogenesis and intraductal epithelial hyperplasia but not lobular differentiation in nulliparous female rats. KGF causes ductal and lobular epithelial hyperplasia in male...

  10. Imunidade inata da glândula mamária bovina: resposta à infecção Innate immunity of the bovine mammary gland: response to infection

    Directory of Open Access Journals (Sweden)

    Deolinda Maria Vieira Filha Carneiro

    2009-09-01

    infection's site by numerous stimulations, and these answers are not enhanced by repeated exposure to the same agent. The first obstacle to be faced by the agent is the barrier represented by the teat sphincter and the keratin plug. When the pathogenic agent crosses the teat canal and reaches the teat cistern, starts to act the humoral factors and the phagocytic cells starts do act. Among the humoral mediators there are the lactoperoxidase, complement, cytokines, lactoferrin, lysozyme and NAGase. The non-specific cellular defenses are represented by neutrophils, macrophages and natural killer cells. If these mechanisms have been functioning adequately, the majority of pathogens will be eliminated in a short time, before the specific immune system be activated. The fast elimination of the microorganisms will not allow these alterations in the amount or quality of produced milk. The best understanding of the defense mechanisms of the mammary gland and its alterations during the critical periods of infection, is an useful tool in devising and developing methods to control the mastitis, the major illness of dairy ruminants. This paper overviewed the most important aspects of the innate immunity of bovine mammary gland.

  11. Clock circadian regulator (CLOCK) gene network expression patterns in bovine adipose, liver, and mammary gland at 3 time points during the transition from pregnancy into lactation.

    Science.gov (United States)

    Wang, M; Zhou, Z; Khan, M J; Gao, J; Loor, J J

    2015-07-01

    The transition from late gestation to early lactation is the most critical phase of the lactation cycle for mammals. Research in rodents has revealed changes in the clock circadian regulator (CLOCK) gene network expression around parturition. However, their expression profiles and putative functions during the periparturient period in ruminants remain to be determined. The present study aimed to investigate the expression pattern of the CLOCK network and selected metabolic genes simultaneously in mammary gland (MG), liver (LIV), and subcutaneous adipose tissue (AT). Seven dairy cows were biopsied at -10 (±2), 7, and 21 d relative to parturition. A day × tissue interaction was observed for ARNTL, CRY1, and PER2 due to upregulation at 7 and 21 d postpartum, with their expression being greater in AT and MG compared with LIV. No interaction was detected for CLOCK, CRY2, PER1, and PER3. In general, the expression of NPAS2, NR1D1, NR2F2, ALAS1, FECH, FBXW11, CCRN4L, PPARA, PPARGC1A, and FGF21 was lower at -10 d but increased postpartum in all tissues. The interaction detected for CSNK1D was associated with increased expression postpartum in AT and MG but not LIV. The interaction detected for CPT1A was due to upregulation in AT and LIV postpartum without a change in MG. In contrast, the interaction for PPARG was due to upregulation in AT and MG postpartum but a downregulation in LIV. Leptin was barely detectable in LIV, but there was an interaction effect in AT and MG associated with upregulation postpartum in MG and downregulation in AT. Together, these results suggest that the control of metabolic adaptations in LIV, MG, and AT around parturition might be partly regulated through the CLOCK gene network. Although the present study did not specifically address rhythmic control of tissue metabolism via the CLOCK gene network, the difference in expression of genes studied among tissues confirms that the behavior of circadian-controlled metabolic genes around parturition

  12. Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells.

    Science.gov (United States)

    Qian, Xi; Zhao, Feng-Qi

    2013-03-01

    Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1. PMID:23313770

  13. Persistence of gamma-H2AX and 53BP1 foci in proliferating and nonproliferating human mammary epithelial cells after exposure to gamma-rays or iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Groesser, Torsten; Chang, Hang; Fontenay, Gerald; Chen, James; Costes, Sylvain V.; Barcellos-Hoff, Mary Helen; Parvin, Bahram; Rydberg, Bjorn

    2010-12-22

    To investigate {gamma}-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionizing radiation under different cell culture conditions. HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced {gamma}-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both {gamma}-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after {gamma}-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. Conclusions: The disappearance of radiation induced {gamma}-H2AX and 53BP1 foci in HMEC have different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent {gamma}-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodeling.

  14. Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis: role of INK4a/CAV-1 in mammary epithelial cell hyperplasia.

    Science.gov (United States)

    Williams, Terence M; Lee, Hyangkyu; Cheung, Michelle W-C; Cohen, Alex W; Razani, Babak; Iyengar, Puneeth; Scherer, Philipp E; Pestell, Richard G; Lisanti, Michael P

    2004-06-01

    Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus. PMID:15044451

  15. Genetic mechanisms in Apc-mediated mammary tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Mari Kuraguchi

    2009-02-01

    Full Text Available Many components of Wnt/beta-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre-mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of beta-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; Apc(CKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre-induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre-mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of beta-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.

  16. Role of JNK in mammary gland development and breast cancer

    OpenAIRE

    Cellurale, Cristina; Girnius, Nomeda; Jiang, Feng; Cavanagh-Kyros, Julie; Lu, Shaolei; Garlick, David S.; Mercurio, Arthur M.; Davis, Roger J

    2011-01-01

    JNK signaling has been implicated in the developmental morphogenesis of epithelial organs. In this study we employed a compound deletion of the murine Jnk1 and Jnk2 genes in the mammary gland to evaluate the requirement for these ubiquitously expressed genes in breast development and tumorigenesis. JNK1/2 was not required for breast epithelial cell proliferation or motility. However, JNK1/2 deficiency caused increased branching morphogenesis and defects in the clearance of lumenal epithelial ...

  17. Loss of vitamin D receptor signaling from the mammary epithelium or adipose tissue alters pubertal glandular development

    OpenAIRE

    Johnson, Abby L.; Zinser, Glendon M.; Waltz, Susan E.

    2014-01-01

    Vitamin D3 receptor (VDR) signaling within the mammary gland regulates various postnatal stages of glandular development, including puberty, pregnancy, involution, and tumorigenesis. Previous studies have shown that vitamin D3 treatment induces cell-autonomous growth inhibition and differentiation of mammary epithelial cells in culture. Furthermore, mammary adipose tissue serves as a depot for vitamin D3 storage, and both epithelial cells and adipocytes are capable of bioactivating vitamin D3...

  18. Lessons Learned from Mouse Mammary Tumor Virus in Animal Models.

    Science.gov (United States)

    Dudley, Jaquelin P; Golovkina, Tatyana V; Ross, Susan R

    2016-03-31

    Mouse mammary tumor virus (MMTV), which was discovered as a milk-transmitted, infectious, cancer-inducing agent in the 1930s, has been used as an animal model for the study of retroviral infection and transmission, antiviral immune responses, and breast cancer and lymphoma biology. The main target cells for MMTV infection in vivo are cells of the immune system and mammary epithelial cells. Although the host mounts an immune response to the virus, MMTV has evolved multiple means of evading this response. MMTV causes mammary tumors when the provirus integrates into the mammary epithelial and lymphoid cell genome during viral replication and thereby activates cellular oncogene expression. Thus, tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer. PMID:27034391

  19. Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

    OpenAIRE

    Rodriguez-Zas Sandra L; Bionaz Massimo; Morin Dawn E; Drackley James K; Moyes Kasey M; Everts Robin E; Lewin Harris A; Loor Juan J

    2009-01-01

    Abstract Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection...

  20. Mammary carcinoma diagnostics and therapy

    International Nuclear Information System (INIS)

    The book on mammary carcinoma diagnostics and therapy covers the following issues: development, anatomy and physiology of the mammary glands, pathology of benign and malign mammary gland changes, non-imaging diagnostics; mammography; ultrasonic mammography; magnetic resonance tomography of the mammary glands; imaging diagnostics findings; mammary interventions; examination concepts; operative therapy of the mammary carcinoma; chemotherapy of the mammary carcinoma; radio-oncological therapy of the mammary carcinoma; logistics in a medical center for mammary gland diseases; logistics in an interdisciplinary center for mammary diseases; dialogue conduction and psycho-social attendance.

  1. Pharmacokinetics and pharmacodynamics of gamithromycin in pulmonary epithelial lining fluid in naturally occurring bovine respiratory disease in multisource commingled feedlot cattle.

    Science.gov (United States)

    DeDonder, K D; Apley, M D; Li, M; Gehring, R; Harhay, D M; Lubbers, B V; White, B J; Capik, S F; KuKanich, B; Riviere, J E; Tessman, R K

    2016-04-01

    The objectives of this study were to determine (i) whether an association exists between individual pharmacokinetic parameters and treatment outcome when feeder cattle were diagnosed with bovine respiratory disease (BRD) and treated with gamithromycin (Zactran(®) ) at the label dose and (ii) whether there was a stronger association between treatment outcome and gamithromycin concentration in plasma or in the pulmonary epithelial lining fluid (PELF) effect compartment. The study design was a prospective, blinded, randomized clinical trial utilizing three groups of 60 (362-592 lb) steers/bulls randomly allocated within origin to sham injection or gamithromycin mass medication. Cattle were evaluated daily for signs of BRD by a veterinarian blinded to treatment. Animals meeting the BRD case definition were enrolled and allocated to a sample collection scheme consisting of samples for bacterial isolation (bronchoalveolar lavage fluid and nasopharyngeal swabs) and gamithromycin concentration determination (PELF and plasma). Gamithromycin susceptibility of M. haemolytica (n = 287) and P. multocida (n = 257) were determined using broth microdilution with frozen panels containing gamithromycin at concentrations from 0.03 to 16 μg/mL. A two-compartment plasma pharmacokinetic model with an additional compartment for gamithromycin in PELF was developed using rich data sets from published and unpublished studies. The sparse data from our study were then fit to this model using nonlinear mixed effects modeling to estimate individual parameter values. The resulting parameter estimates were used to simulate full time-concentration profiles for each animal in this study. These profiles were analyzed using noncompartmental methods so that PK/PD indices (AUC24 /MIC, AUC∞ /MIC, CMAX /MIC) could be calculated for plasma and PELF (also T>MIC) for each individual. The calculated PK/PD indices were indicative that for both M. haemolytica and P. multocida a higher drug

  2. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    OpenAIRE

    Streuli, Charles H

    2011-01-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional di...

  3. Signalling pathways implicated in early mammary gland morphogenesis and breast cancer.

    Directory of Open Access Journals (Sweden)

    Beatrice Howard

    2006-08-01

    Full Text Available Specification of mammary epithelial cell fate occurs during embryogenesis as cells aggregate to form the mammary anlage. Within the embryonic mammary bud, a population of epithelial cells exists that will subsequently proliferate to form a ductal tree filling the stromal compartment, and which can produce milk upon terminal differentiation after birth. Subsequently, these structures can be remodelled and returned to a basal state after weaning before regenerating in future pregnancies. The plasticity of the mammary epithelial cell, and its responsiveness to hormone receptors, facilitates this amazing biological feat, but aberrant signalling may also result in unintended consequences in the form of frequent malignancies. Reflecting this intimate connection, a considerable number of signalling pathways have been implicated in both mammary gland morphogenesis and carcinogenesis.

  4. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Lixin Liu

    2015-07-01

    Full Text Available As a protective factor for lipopolysaccharide (LPS-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, interleukin-1β (IL-1β and inducible nitric oxide synthase (iNOS. Enzyme-linked immunosorbent assay (ELISA analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB and mitogen-activated protein kinase (MAPKs and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK, p38 mitogen-activated protein kinase (p38MAPK and inhibitor of NF-κB (IκB phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR, ribosomal protein S6 kinase 1 (S6K1, serine/threonine protein kinase Akt 1 (AKT1, sterol regulatory element binding protein 1 (SREBP1 and peroxisome proliferator-activated receptor gamma

  5. Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture

    OpenAIRE

    Talhouk, Rabih S.; CHIN, JENNIE R.; UNEMORI, ELAINE N.; Werb, Zena; Bissell, Mina J.

    1991-01-01

    The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradat...

  6. Adipophilin regulates maturation of cytoplasmic lipid droplets and alveolae in differentiating mammary glands

    OpenAIRE

    Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Palmer, Carol; Chang, Benny Hung-Junn; Chan, Lawrence; McManaman, James L.

    2011-01-01

    Milk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD fo...

  7. Loss of sfrp1 promotes ductal branching in the murine mammary gland

    OpenAIRE

    Gauger Kelly J; Shimono Akihiko; Crisi Giovanna M; Schneider Sallie

    2012-01-01

    Abstract Background Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics obs...

  8. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    OpenAIRE

    Lund, Leif R.; Rømer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J.; Danø, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when ...

  9. Lineage associated expression of virulence traits in bovine-adapted Staphylococcus aureus.

    Science.gov (United States)

    Budd, Kathleen E; Mitchell, Jennifer; Keane, Orla M

    2016-06-30

    Bovine mastitis is the most costly disease to the dairy industry worldwide with Staphylococcus aureus commonly associated with intramammary infections that are persistent and refractory to treatment. The strains of S. aureus that cause mastitis predominantly belong to a number of well-described bovine-adapted lineages. The objective of this study was to determine if a variety of potential virulence traits were associated with lineage. Bovine-adapted S. aureus isolates (n=120), belonging to lineages CC97, CC151 and ST136, were tested for their ability to adhere to and internalise within cultured bovine mammary epithelial cells (bMEC), to bind bovine fibronectin, to form a biofilm in TSB, TSB+1% glucose and TSB+4% NaCl, and to induce an immune response from bMEC. There were no significant differences between the lineages in ability to adhere to or internalise within bMEC although there were significant differences between individual isolates. For lineages CC97 and ST136, mammalian cell adherence was correlated with the ability to bind bovine fibronectin, however isolates from CC151 could not bind bovine fibronectin in vitro, but adhered to bMEC in a fibronectin-independent manner. There were significant differences between the lineages in ability to form a biofilm in all three growth media with ST136 forming the strongest biofilm while CC151 formed the weakest biofilm. Lineages also differed in their ability to elicit an immune response from bMEC with CC97 eliciting a stronger immune response than CC151 and ST136. These data indicate the potential for both lineage and strain-specific virulence and a strain-specific response to infection in vivo and caution against extrapolating an effect from a single strain of S. aureus to draw conclusions regarding virulence or the host response to infection in unrelated lineages. PMID:27259823

  10. BOVINE VIRAL DIARRHEA VIRUSES

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) is an umbrella term for two species of viruses, BVDV1 and BVDV2, within the Pestivirus genus of the Flavivirus family. BVDV viruses are further subclassified as cytopathic and noncytopathic based on their activity in cultured epithelial cells. Noncytopathic BVDV p...

  11. β1 integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli

    OpenAIRE

    Li, Na; Zhang, Yu; Naylor, Matthew J.; Schatzmann, Franziska; Maurer, Francisca; Wintermantel, Tim; Schuetz, Gunther; Mueller, Ulrich; Streuli, Charles H; Hynes, Nancy E.

    2005-01-01

    Integrin–extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of β1 integrin in the mammary gland, Itgβ1flox/flox mice were crossed with WAPiCre transgenic mice, which led to specific ablation of β1 integrin in luminal alveolar epithelial cells. In the β1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell–basement membr...

  12. Mammary stem cells: Novel markers and novel approaches to increase lactation efficiency

    Science.gov (United States)

    Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue r...

  13. Signal Transducer and Activator of Transcription 5a Mediates Mammary Ductal Branching and Proliferation in the Nulliparous Mouse

    OpenAIRE

    Santos, Sarah J.; Haslam, Sandra Z.; Conrad, Susan E.

    2010-01-01

    Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a−/− mice. The Stat5a-null mammary ...

  14. Immunoglobins in mammary secretions

    DEFF Research Database (Denmark)

    Hurley, W L; Theil, Peter Kappel

    2013-01-01

    Immunoglobulins secreted in colostrum and milk by the lactating mammal are major factors providing immune protection to the newborn. Immunoglobulins in mammary secretions represent the cumulative immune response of the lactating animal to exposure to antigenic stimulation that occurs through...... immunoglobulins found in mammary secretions in the context of their diversity of structure, origin, mechanisms of transfer, and function....

  15. PKA signaling drives mammary tumorigenesis through Src.

    Science.gov (United States)

    Beristain, A G; Molyneux, S D; Joshi, P A; Pomroy, N C; Di Grappa, M A; Chang, M C; Kirschner, L S; Privé, G G; Pujana, M A; Khokha, R

    2015-02-26

    Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans. PMID:24662820

  16. Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development.

    Science.gov (United States)

    Tan, J; Buache, E; Alpy, F; Daguenet, E; Tomasetto, C-L; Ren, G-S; Rio, M-C

    2014-07-31

    MMP-11 is a bad prognosis paracrine factor in invasive breast cancers. However, its mammary physiological function remains largely unknown. In the present study we have investigated MMP-11 function during postnatal mammary gland development and function using MMP-11-deficient (MMP-11-/-) mice. Histological and immunohistochemical analyses as well as whole-mount mammary gland staining show alteration of the mammary gland in the absence of MMP-11, where ductal tree, alveolar structures and milk production are reduced. Moreover, a series of transplantation experiments allowed us to demonstrate that MMP-11 exerts an essential local paracrine function that favors mammary gland branching and epithelial cell outgrowth and invasion through adjacent connective tissues. Indeed, MMP-11-/- cleared fat pads are not permissive for wild-type epithelium development, whereas MMP-11-/- epithelium transplants grow normally when implanted in wild-type cleared fat pads. In addition, using primary mammary epithelial organoids, we show in vitro that this MMP-11 pro-branching effect is not direct, suggesting that MMP-11 acts via production/release of stroma-associated soluble factor(s). Finally, the lack of MMP-11 leads to decreased periductal collagen content, suggesting that MMP-11 has a role in collagen homeostasis. Thus, local stromal MMP-11 might also regulate mammary epithelial cell behavior mechanically by promoting extracellular matrix stiffness. Collectively, the present data indicate that MMP-11 is a paracrine factor involved during postnatal mammary gland morphogenesis, and support the concept that the stroma strongly impact epithelial cell behavior. Interestingly, stromal MMP-11 has previously been reported to favor malignant epithelial cell survival and promote cancer aggressiveness. Thus, MMP-11 has a paracrine function during mammary gland development that might be harnessed to promote tumor progression, exposing a new link between development and malignancy. PMID:24141782

  17. Control of ductal vs. alveolar differentiation of mammary clonogens and susceptibility to radiation-induced mammary cancer

    International Nuclear Information System (INIS)

    We have developed an in vitro-in vivo transplantation assay for measuring the concentration of clonogenic epithelial cells in cell suspensions of rat mammary tissue. Rat mammary clonogens from organoid cultures are capable of the same degree of PLDR as clonogens in vivo. The growth and differentiation of mammary clonogens to alveolar colonies or ductal colonies is regulated as follows: a) in the presence of E2 and high prolactin (Prl), cortisol induces mammary clonogens to proliferate and differentiate to form alveolar colonies which secrete milk and begin losing clonogenic potential, b) in cortisol deficient rats, Prl and E2 synergistically stimulate non-secretory ductal colonies, formation of which retain clonogenic potential, c) E2 without progesterone stimulates alveolar colony formation in the presence of cortical and high Prl, d) progesterone inhibits mammary clonogen differentiation to milk-producing cells and induces ductogenesis in a dose responsive fashion in the presence of E2, cortisol and high Prl. High prolactin levels coupled with glucocorticoid deficiency increases the susceptibility to mammary carcinogenesis following low dose radiation exposure by increasing the number of total mammary clonogens which are the presumptive target cells and by stimulating their proliferation after exposure. (author)

  18. Prolactin Suppression of Gonadotropin-Releasing Hormone Initiation of Mammary Gland Involution in Female Rats.

    Science.gov (United States)

    Rieanrakwong, Duangjai; Laoharatchatathanin, Titaree; Terashima, Ryota; Yonezawa, Tomohiro; Kurusu, Shiro; Hasegawa, Yoshihisa; Kawaminami, Mitsumori

    2016-07-01

    It has been demonstrated that mammary gland involution after lactation is initiated by accumulation of milk in alveoli after weaning. Here, we report that involution is also dependent on mammary GnRH expression that is suppressed by PRL during lactation. Reduction of plasma prolactin (PRL) by the withdrawal of suckling stimuli increased GnRH and annexin A5 (ANXA5) expression in the mammary tissues after lactation with augmentation of epithelial apoptosis. Intramammary injection of a GnRH antagonist suppressed ANXA5 expression and apoptosis of epithelial cells after forcible weaning at midlactation, whereas local administration of GnRH agonist (GnRHa) caused apoptosis of epithelial cells with ANXA5 augmentation in lactating rats. The latter treatment also decreased mammary weight, milk production, and casein accumulation. Mammary mast cells were strongly immunopositive for GnRH and the number increased in the mammary tissues after weaning. GnRHa was shown to be a chemoattractant for mast cells by mammary local administration of GnRHa and Boyden chamber assay. PRL suppressed the mammary expression of both ANXA5 and GnRH mRNA. It also decreased mast cell numbers in the gland after lactation. These results are the first to demonstrate that GnRH, synthesized locally in the mammary tissues, is required for mammary involution after lactation. GnRH is also suggested to introduce mast cells into the regressing mammary gland and would be in favor of tissue remodeling. The suppression of these processes by PRL is a novel physiological function of PRL. PMID:27175971

  19. Unique expression pattern of the three insulin receptor family members in the rat mammary gland

    DEFF Research Database (Denmark)

    Hvid, Henning; Klopfleisch, Robert; Vienberg, Sara Gry; Hansen, Bo F.; Thorup, Inger; Jensen, Henrik Elvang; Oleksiewicz, Martin B.

    2011-01-01

    mammary gland. Using laser micro-dissection, quantitative RT-PCR and immunohistochemistry, we examined the expression of IR (insulin receptor), IGF-1R (IGF-1 receptor), IRR (insulin receptor-related receptor), ERα (estrogen receptor alpha), ERβ (estrogen receptor beta) and PR (progesteron receptor) in...... young, virgin, female Sprague-Dawley rats and compared to expression in reference organs. The mammary gland displayed the highest expression of IRR and IGF-1R. In contrast, low expression of IR transcripts was observed in the mammary gland tissue with expression of the IR-A isoform being 5-fold higher...... than the expression of the IR-B. By immunohistochemistry, expression of IR and IGF-1R was detected in all mammary gland epithelial cells. Expression of ERα and PR was comparable between mammary gland and ovary, whereas expression of ERβ was lower in mammary gland than in the ovary. Finally, expression...

  20. In vitro adherence patterns of Shigella serogroups to bovine recto-anal junction squamous epithelial (RSE) cells are similar to those of Escherichia coli O157

    Science.gov (United States)

    The aim of this study was to determine whether Shigella species, which are human gastrointestinal pathogens, can adhere to cattle recto-anal junction squamous epithelial (RSE) cells using a recently standardized adherence assay, and to compare their adherence patterns to that of Escherichia coli O15...

  1. Tissue proteomics of the human mammary gland

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Cabezón, Teresa; Gromova, Irina;

    2010-01-01

    phenotypes of the different cell subpopulations present in normal human mammary tissue, partly due to the formidable heterogeneity of mammary tissue, but also due to limitations of the current proteomic technologies. Work in our laboratories has attempted to address in a systematic fashion some of these...... biomarker discovery program. We review and present new data on the putative cell-progenitor marker cytokeratin 15 (CK15), and describe a novel marker, dihydropyriminidase-related protein 3 (DRP3) that in combination with CK15 and other well known proteins were used to define molecular phenotypes of normal...... human breast epithelial cells and their progenitors in resting acini, lactating alveoli, and large collecting ducts of the nipple. Preliminary results are also presented concerning DRP3 positive usual ductal hyperplasias (UDHs) and on single cell layer columnar cells (CCCs). At least two bona fide...

  2. Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

    Directory of Open Access Journals (Sweden)

    Rodriguez-Zas Sandra L

    2009-11-01

    Full Text Available Abstract Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (IMI with S. uberis and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with S. uberis O140J. Results Streptococcus uberis IMI resulted in 2,102 (1,939 annotated differentially expressed genes (DEG. Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each, the majority of which were signaling pathways. Among the most inhibited were LXR/RXR Signaling and PPARα/RXRα Signaling. Pathways activated by IMI were IL-10 Signaling and IL-6 Signaling which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA3. Genes down-regulated (1,020 included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Network analysis of DEG indicated that TNF had positive relationships with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2 and negative relationships with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL and antioxidant activity (SOD1. Conclusion Results provided novel information into the early signaling and metabolic pathways in mammary tissue that are associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J elicited

  3. Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell profileration and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

    DEFF Research Database (Denmark)

    Moyes, Kasey M; Drackley, James K; Morin, Dawn E;

    2009-01-01

    Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and....../or metabolic responses to mastitis challenge with S. uberis O140J. Results Streptococcus uberis IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (DEG). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were...

  4. Nuclear repartitioning of galectin-1 by an extracellular glycan switch regulates mammary morphogenesis.

    Science.gov (United States)

    Bhat, Ramray; Belardi, Brian; Mori, Hidetoshi; Kuo, Peiwen; Tam, Andrew; Hines, William C; Le, Quynh-Thu; Bertozzi, Carolyn R; Bissell, Mina J

    2016-08-16

    Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Here we show that galectin-1 (Gal-1), an endogenous lectin that recognizes glycans bearing N-acetyllactosamine (LacNAc) epitopes, induces branching migration of mammary epithelia in vivo, ex vivo, and in 3D organotypic cultures. Surprisingly, Gal-1's effects on mammary patterning were independent of its glycan-binding ability and instead required localization within the nuclei of mammary epithelia. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Specifically, α2,6-sialylation of terminal LacNAc residues in the end buds masked Gal-1 ligands, thereby liberating the protein for nuclear translocation. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. We also found that malignant breast cells possess higher levels of nuclear Gal-1 and α2,6-SA and lower levels of LacNAc than nonmalignant cells in culture and in vivo and that nuclear localization of Gal-1 promotes a transformed phenotype. Our findings suggest that differential glycosylation at the level of tissue microanatomy regulates the nuclear function of Gal-1 in the context of mammary gland morphogenesis and in cancer progression. PMID:27496330

  5. Redirection of Human Cancer Cells upon the Interaction with the Regenerating Mouse Mammary Gland Microenvironment

    Directory of Open Access Journals (Sweden)

    Sonia M. Rosenfield

    2013-01-01

    Full Text Available Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo.

  6. Lgr5-Expressing Cells Are Sufficient and Necessary for Postnatal Mammary Gland Organogenesis

    Directory of Open Access Journals (Sweden)

    Vicki Plaks

    2013-01-01

    Full Text Available Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5+ mammary epithelial cells (MECs. We found that Lgr5+ cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs, are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5+ cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.

  7. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis.

    Science.gov (United States)

    Plaks, Vicki; Brenot, Audrey; Lawson, Devon A; Linnemann, Jelena R; Van Kappel, Eline C; Wong, Karren C; de Sauvage, Frederic; Klein, Ophir D; Werb, Zena

    2013-01-31

    Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5(+)) mammary epithelial cells (MECs). We found that Lgr5(+) cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5(+) cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis. PMID:23352663

  8. Expression and function of leptin and its receptor in mouse mammary gland

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lactation- and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.

  9. Wnt4 is not sufficient to induce lobuloalveolar mammary development

    Directory of Open Access Journals (Sweden)

    Pelegri Francisco

    2009-10-01

    Full Text Available Abstract Background Brisken et al (2000 showed that Wnt4 null mammary glands were deficient in early lobuloalveolar mammary outgrowth during pregnancy, and implicated Wnt4 as an effector for the progesterone-induced mammary growth program. Though ectopic Wnt1 signaling is known to be mitogenic and oncogenic, no endogenously expressed Wnt ligands have ever been directly implicated in mammary growth and morphogenesis. Therefore, we generated conditional transgenic mice to test whether Wnt4 can stimulate mammary epithelial cell growth. Results We found that despite pregnancy-associated expression levels of Wnt4, mammary glands did not display the side-branching typical of early pregnancy. Control experiments designed to test the Wnt4 construct in zebrafish reproduced other studies that demonstrated Wnt4-specific phenotypes distinct from Wnt1-induced phenotypes. Indeed, using qPCR-based array analyses, we found that a specific transcriptional target of Wnt4, namely Wnt16, was induced in Wnt4-expressing transgenic glands, to levels equivalent to that of early pregnant glands. Conclusion Taken together, we propose that Wnt4 is necessary, but not sufficient, to induce side-branch development.

  10. Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

    Directory of Open Access Journals (Sweden)

    Rebecca Cote

    Full Text Available Shiga toxin-producing Escherichia coli O157:H7 (O157 are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ. Some colonized animals, referred to as "super-shedders" (SS, are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces. Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17 revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359, including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

  11. Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

    OpenAIRE

    Zheng, L; Zhang, S.; W. Xue; Kapil, S; Minocha, H C

    1998-01-01

    The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demo...

  12. Tubulopapillary carcinoma of the mammary gland in a maned wolf (Chrysocyon brachyurus: histopathological and immunophenotypical analysis

    Directory of Open Access Journals (Sweden)

    C.O. Gamba

    2011-12-01

    Full Text Available A maned female wolf (Chrysocyon brachyurus showed nodules in the inguinal and left abdominal cranial mammary glands. The mammary gland was surgically excised, and microscopic analysis revealed epithelial cell proliferation in a tubular and papillary pattern; delicate fibrovascular stalks presenting numerous layers of moderately pleomorfic epithelial cells were observed. This histologic appearance was compatible with a diagnosis of mammary tubulopapillary carcinoma. The immunohistochemical profile revealed nuclear positivity for estrogen (70% and progesterone (at least 90% of the neoplastic cells. The myoepithelium-associated with neoplastic cells lacked integrity, as evidenced by failed smooth muscle alpha actin reactivity in microinvasive areas. A low proliferation index was observed (3.4%. To the authors' knowledge, the present case represents the first finding of female tubulopapillary carcinoma in a mammary gland in this species.

  13. Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors

    International Nuclear Information System (INIS)

    Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral

  14. Correlation of hypothetical virulence traits of two Streptococcus uberis strains with the clinical manifestation of bovine mastitis.

    Science.gov (United States)

    Tassi, Riccardo; McNeilly, Tom N; Sipka, Anja; Zadoks, Ruth N

    2015-01-01

    Streptococcus uberis is a common cause of clinical and subclinical mastitis in dairy cattle. Several virulence mechanisms have been proposed to contribute to the species' ability to cause disease. Here, virulence characteristics were compared between S. uberis strains FSL Z1-048, which consistently caused clinical mastitis in a challenge model, and FSL Z1-124, which consistently failed to cause disease in the same model, to ascertain whether in vitro virulence characteristics were related to clinical outcome. Macrophages derived from bovine blood monocytes failed to kill FSL Z1-048 whilst reducing survival of FSL Z1-124 by 42.5%. Conversely, blood derived polymorphonuclear cells caused more reduction (67.1 vs. 44.2%, respectively) in the survival of FSL Z1-048 than in survival of FSL Z1-124. After 3 h of coincubation with bovine mammary epithelial cell line BME-UV1, 1000-fold higher adherence was observed for FSL Z1-048 compared to FSL Z1-124, despite presence of a frame shift mutation in the sua gene of FSL Z1-048 that resulted in predicted truncation of the S. uberis Adhesion Molecule (SUAM) protein. In contrast, FSL Z1-124 showed higher ability than FSL Z1-048 to invade BME-UV1 cells. Finally, observed biofilm formation by FSL Z1-124 was significantly greater than for FSL Z1-048. In summary, for several hypothetical virulence characteristics, virulence phenotype in vitro did not match disease phenotype in vivo. Evasion of macrophage killing and adhesion to mammary epithelial cells were the only in vitro traits associated with virulence in vivo, making them attractive targets for further research into pathogenesis and control of S. uberis mastitis. PMID:26497306

  15. Metabolomic Changes Accompanying Transformation and Acquisition of Metastatic Potential in a Syngeneic Mouse Mammary Tumor Model*

    OpenAIRE

    Lu, Xin; Bennet, Bryson; Mu, Euphemia; Rabinowitz, Joshua; Kang, Yibin

    2010-01-01

    Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increas...

  16. Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

    OpenAIRE

    Zvelebil, Marketa; Oliemuller, Erik; Gao, Qiong; Wansbury, Olivia; Mackay, Alan; Kendrick, Howard; Matthew J Smalley; Reis-Filho, Jorge S.; Howard, Beatrice A

    2013-01-01

    Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with ...

  17. An Essential Role for Cdc42 in the Functioning of the Adult Mammary Gland.

    Science.gov (United States)

    Druso, Joseph E; Endo, Makoto; Lin, Miao-Chong Joy; Peng, Xu; Antonyak, Marc A; Meller, Stephanie; Cerione, Richard A

    2016-04-22

    The Rho family small GTPase Cdc42 has been implicated in a wide range of cellular functions including the establishment of cell polarity and the remodeling of the actin cytoskeletal architecture, resulting in the tight regulation of cell growth and survival during developmental processes. The complete knock-out of Cdc42 in the mouse is embryonic-lethal, and its targeted deletion in various tissues has been shown to disrupt tissue homeostasis. Thus far, in most studies, the targeted deletion of Cdc42 occurred during embryogenesis. Here, we have used a conditional gene deletion strategy in mice to probe the specific role of Cdc42 during adult mammary gland function. Cdc42 conditional-knock-out females were unable to adequately nourish their pups, due to a disorganized epithelial compartment within their mammary glands. A closer examination showed that their mammary epithelial cells were not able to maintain functional alveolar lumens, due to an inability to establish normal apical/basal epithelial polarity, as well as proper cell-cell contacts. Loss of these essential epithelial characteristics led to a premature sloughing off of the Cdc42-null epithelial cells. Overall our findings demonstrate that Cdc42 plays essential roles in mammary gland function post pregnancy, where it helps to establish proper epithelial cell polarity and tissue homeostasis during lactation. PMID:26912661

  18. A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

    Science.gov (United States)

    Song, Jiazhe; Xue, Kai; She, Ji; Ding, Fangrong; Li, Song; Shangguan, Rulan; Dai, Yunping; Du, Liying; Li, Ning

    2014-09-10

    The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment. PMID:24954407

  19. Loss of sfrp1 promotes ductal branching in the murine mammary gland

    Directory of Open Access Journals (Sweden)

    Gauger Kelly J

    2012-08-01

    Full Text Available Abstract Background Secreted frizzled-related proteins (SFRPs are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer stem-like cells. However, the effects of SFRP1 loss on mammary gland development in vivo are yet to be elucidated. The work described here was initiated to investigate the role of SFRP1 in mammary gland development and whether SFRP1−/− mice exhibit changes in mammary gland morphology and cell signaling pathways shown to be associated with SFRP1 loss in vitro. Results 10 week old nulliparous SFRP1−/− mammary glands exhibited branching with clear lobulo-alveolar development, which normally only occurs in hormonally stimulated mid-pregnant wt mammary glands. Explant cultures of SFRP1−/− mammary glands display increased levels of a well known Wnt signaling target gene, Axin2. Histomorphologic evaluation of virgin glands revealed that by 10 weeks of age, the duct profile is markedly altered in SFRP1−/− mice showing a significantly higher density of ducts with distinct alveoli present throughout the mammary gland, and with focal ductal epithelial hyperplasia. These findings persist as the mice age and are evident at 23 weeks of age. Changes in gene expression, including c-Myc, TGFβ-2, Wnt4, RANKL, and Rspo2 early in mammary gland development are consistent with the excessive hyper branching phenotype. Finally, we found that loss of SFRP1 significantly increases the number of mammary epithelial cells capable of mammosphere formation. Conclusions Our study indicates that SFRP1 gene is critical for maintaining proper

  20. Keratinocyte Growth Factor Causes Cystic Dilation of the Mammary Glands of Mice: Interactions of Keratinocyte Growth Factor, Estrogen, and Progesterone In Vivo

    OpenAIRE

    Yi, Eunhee S; Bedoya, Adriana A.; Lee, HyeSun; Kim, Seokhyun; Housley, Regina M.; Aukerman, Sharon L.; Tarpley, John E.; Starnes, Charles; Yin, Songmei; Pierce, Glenn F.; Ulich, Thomas R.

    1994-01-01

    Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically n...

  1. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    Science.gov (United States)

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

    2016-06-01

    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene. PMID:26197710

  2. Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

    Science.gov (United States)

    Anand, Vijay; Jaswal, Shalini; Singh, Surender; Kumar, Sudarshan; Jena, Manoj Kumar; Verma, Arvind Kumar; Yadav, Munna Lal; Janjanam, Jagadeesh; Lotfan, Masoud; Malakar, Dhruba; Dang, Ajay Kumar; Mohanty, Tushar Kumar; Kaushik, Jai Kumar; Mohanty, Ashok Kumar

    2016-02-01

    MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented. PMID:26659075

  3. Proteotypic classification of spontaneous and transgenic mammary neoplasms

    International Nuclear Information System (INIS)

    Mammary tumors in mice are categorized by using morphologic and architectural criteria. Immunolabeling for terminal differentiation markers was compared among a variety of mouse mammary neoplasms because expression of terminal differentiation markers, and especially of keratins, provides important information on the origin of neoplastic cells and their degree of differentiation. Expression patterns for terminal differentiation markers were used to characterize tumor types and to study tumor progression in transgenic mouse models of mammary neoplasia (mice overexpressing Neu (Erbb2), Hras, Myc, Notch4, SV40-TAg, Tgfa, and Wnt1), in spontaneous mammary carcinomas, and in mammary neoplasms associated with infection by the mouse mammary tumor virus (MMTV). On the basis of the expression of terminal differentiation markers, three types of neoplasm were identified: first, simple carcinomas composed exclusively of cells with a luminal phenotype are characteristic of neoplasms arising in mice transgenic for Neu, Hras, Myc, Notch4, and SV40-TAg; second, 'complex carcinomas' displaying luminal and myoepithelial differentiation are characteristic of type P tumors arising in mice transgenic for Wnt1, neoplasms arising in mice infected by the MMTV, and spontaneous adenosquamous carcinomas; and third, 'carcinomas with epithelial to mesenchymal transition (EMT)' are a characteristic feature of tumor progression in Hras-, Myc-, and SV40-TAg-induced mammary neoplasms and PL/J and SJL/J mouse strains, and display de novo expression of myoepithelial and mesenchymal cell markers. In sharp contrast, EMT was not detected in papillary adenocarcinomas arising in BALB/cJ mice, spontaneous adenoacanthomas, neoplasms associated with MMTV-infection, or in neoplasms arising in mice transgenic for Neu and Wnt1. Immunohistochemical profiles of complex neoplasms are consistent with a stem cell origin, whereas simple carcinomas might originate from a cell committed to the

  4. Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

    OpenAIRE

    Zhou, Jiong; Chehab, Renee; Tkalcevic, Josephine; Naylor, Matthew J.; Harris, Jessica; Wilson, Trevor J; Tsao, Sue; Tellis, Irene; Zavarsek, Silva; Xu, Dakang; Lapinskas, Erika J; Visvader, Jane; Lindeman, Geoffrey J; Thomas, Ross; Ormandy, Christopher J.

    2005-01-01

    Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blo...

  5. Tumour-stromal interactions: Integrins and cell adhesions as modulators of mammary cell survival and transformation

    International Nuclear Information System (INIS)

    Stromal–epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization. Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease. Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment. Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance

  6. Mammary Glands: Developmental Changes

    Science.gov (United States)

    The mammary gland progresses from the accumulation of a few cells in the embryonic ectoderm to a highly arborescent tubulo-alveolar gland capable of secreting a highly nutritious product for consumption. Throughout this progression, various changes occur during each developmental stage: prenatal, pr...

  7. Radiogenic neoplasia in thyroid and mammary clonogens

    International Nuclear Information System (INIS)

    We have developed rat thyroid and mammary clonogen transplantation systems for the study of radiogenic cancer induction at the target cell level in vivo. The epithelial cell populations of both glands contain small subpopulations of cells which are capable of giving rise to monoclonal glandular structures when transplanted and stimulated with appropriate hormones. During the end of the last grant year and the first half of the current grant year, we have completed analyses and summarized for publication: investigations on the relationship between grafted thyroid cell number and the rapidity and degree of reestablishment of the thyroid-hypothalamicpituitary axis in thyroidectomized rats maintained on a normal diet or an iodine deficient diet; studies of the persistence of, and the differentiation potential and functional characteristics of, the TSH- (thyrotropin-) responsive sub-population of clonogens during goitrogenesis, the plateau-phase of goiter growth, and goiter involution; studies of changes in the size of the clonogen sub-population during goitrogenesis, goiter involution and the response to goitrogen rechallenge; and the results of the large carcinogenesis experiment on the nature of the grafted thyroid cell number-dependent suppression of promotion/progression to neoplasia in grafts of radiation-initiated thyroid cells. We are testing new techniques for the culture, cytofluorescent analysis and characterization mammary epithelial cells and of clonogens in a parallel project, and plan to apply similar technology to the thyroid epithelial cells and clonogen population. Data from these studies will be used in the design of future carcinogenesis experiments on neoplastic initiation by high and low LET radiations and on cells interactions during the neoplastic process

  8. Lactobacillus rhamnosus GR-1 Limits Escherichia coli-Induced Inflammatory Responses via Attenuating MyD88-Dependent and MyD88-Independent Pathway Activation in Bovine Endometrial Epithelial Cells.

    Science.gov (United States)

    Liu, Mingchao; Wu, Qiong; Wang, Mengling; Fu, Yunhe; Wang, Jiufeng

    2016-08-01

    Intrauterine Escherichia coli infection after calving reduces fertility and causes major economic losses in the dairy industry. We investigated the protective effect of the probiotic Lactobacillus rhamnosus GR-1 on E. coli-induced cell damage and inflammation in primary bovine endometrial epithelial cells (BEECs). L. rhamnosus GR-1 reduced ultrastructure alterations and the percentage of BEECs apoptosis after E. coli challenge. Increased messenger RNA (mRNA) expression of immune response indicators, including pattern recognition receptors (toll-like receptor [TLR]2, TLR4, nucleotide-binding oligomerization domain [NOD]1, and NOD2), inflammasome proteins (NOD-like receptor family member pyrin domain-containing protein 3, apoptosis-associated speck-like protein, and caspase-1), TLR4 downstream adaptor molecules (myeloid differentiation antigen 88 [MyD88], toll-like receptor adaptor molecule 2 [TICAM2]), nuclear transcription factor kB (NF-kB), and the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-18, and interferon (IFN)-β, was observed following E. coli challenge. However, these increases were attenuated by L. rhamnosus GR-1 pretreatment. Our data indicate that L. rhamnosus GR-1 ameliorates the E. coli-induced disruption of cellular ultrastructure, subsequently reducing the percentage of BEECs apoptosis and limiting inflammatory responses, partly via attenuation of MyD88-dependent and MyD88-independent pathway activation. Certain probiotics could potentially prevent postpartum uterine diseases in dairy cows, ultimately reducing the use of antibiotics. PMID:27236308

  9. Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection

    OpenAIRE

    Kirchhoff, Jana; Uhlenbruck, Sabine; Goris, Katherina; Keil, Günther M.; Herrler, Georg

    2014-01-01

    Bovine respiratory disease complex (BRDC) is the major cause of serious respiratory tract infections in calves. The disease is multifactorial, with either stress or reduced immunity allowing several pathogens to emerge. We investigated the susceptibility of bovine airway epithelial cells (BAEC) to infection by the three major viruses associated with the BRDC: bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and bovine parainfluenza virus type 3 (BPIV3). For this pu...

  10. Twist基因逆转录病毒载体的构建及其诱导正常乳腺上皮细胞发生上皮间质转化%Construction of retroviral vector carrying Twist gene and its induction of epithelialmesenchymal transition in human mammary epithelial cells

    Institute of Scientific and Technical Information of China (English)

    杨佳佳; 胡萍; 周明莉; 黄杰涛; 柳满然

    2013-01-01

    Objective To construct a retroviral vector carrying Twist gene and investigate its effect on human mammary MCF10A epithelial cells.Methods Myc-Twist was digested from pcDNA3/myc-Twist and subcloned into the retroviral vector pBABE-puro to construct a recombinant plasmid (pBABE-myc-Twist).The inserted Twist gene was confirmed by restriction enzyme digestion and DNA sequencing.The plasmid pBABE-myc-Twist and the packaging plasmid pAmpho were co-transfected into HEK293T cells for packaging of retrovirus.Meanwhile,the control plasmid pBABE-puro and the packaging plasmid were co-transfected into the other HEK293T cells as a control group.Human mammary MCF10A epithelial cells were infected with the retroviruses carrying Twist gene or the controls,and selected by puromycin.The expression of Twist in the MCF10A-Twist and MCF10A-Vector cells was determined by RT-PCR and Westem blotting.The expressions of epithelial-mesenchymal transition (EMT) marker proteins induced by Twist in MCF10A cells were detected using immunofluorescence cytochemistry and Westem blotting.Cell migration and invasion abilities were analyzed by Transwell(R) assay.Results The myc-tagged Twist gene was correctly inserted into the retroviral expression vector as a recombinant plasmid (pBABE-myc-Twist) as identified by restriction analysis and DNA sequencing.The Twist gene was efficiently delivered into human mammary MCF10A epithelial cells by the retrovirus,resulting in the stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT-PCR and Western blotting,respectively.The expression of the epithelial biomarker E-cadherin was downregulated whereas,the mesenchymal marker vimentin upregulated in MCF10A-Twist cells as shown by immunofluorescence cytochemistry and Western blotting.Cell migration and invasion abilities were enhanced notably in MCF10A-Twist cells as compared with MCF10A-Vector control cells (P <0.01).Conclusion Twist induces EMT of MCF10A cells and plays an important role in

  11. Canine mammary tumors - clinical survey

    OpenAIRE

    Elena Atanaskova Petrov; Ksenija Ilievska; Plamen Trojacanec; Irena Celeska; Goran Nikolovski; Ivica Gjurovski; Toni Dovenski

    2014-01-01

    Mammary tumours are the second most frequent neoplasia in dogs, mainly affecting older female patients. Approximately 50% of the mammary tumours are malignant with high percentage of mortality if not treated in time. The aim of this study was to analyze the data of canine patients with mammary tumours, to evaluate the type of tumours, as well as the relationship between tumour incidence and dogs’ age, reproductive cycle and sterilization. The survey was used to retrieve the information in the...

  12. Association of cellular and molecular responses in the rat mammary gland to 17β-estradiol with susceptibility to mammary cancer

    International Nuclear Information System (INIS)

    We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer. Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy. The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed. We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in

  13. Progesterone induces adult mammary stem cell expansion.

    Science.gov (United States)

    Joshi, Purna A; Jackson, Hartland W; Beristain, Alexander G; Di Grappa, Marco A; Mote, Patricia A; Clarke, Christine L; Stingl, John; Waterhouse, Paul D; Khokha, Rama

    2010-06-10

    Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer. PMID:20445538

  14. Myoepithelial Cell Contraction and Milk Ejection Are Impaired in Mammary Glands of Mice Lacking Smooth Muscle Alpha-Actin1

    OpenAIRE

    Haaksma, Carol J.; Schwartz, Robert J.; Tomasek, James J.

    2011-01-01

    Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contr...

  15. Emerging evidence of the physiological role of hypoxia in mammary development and lactation

    Institute of Scientific and Technical Information of China (English)

    Yong Shao; Feng-Qi Zhao

    2014-01-01

    Hypoxia is a physiological or pathological condition of a deficiency of oxygen supply in the body as a whole or within a tissue. During hypoxia, tissues undergo a series of physiological responses to defend themselves against a low oxygen supply, including increased angiogenesis, erythropoiesis, and glucose uptake. The effects of hypoxia are mainly mediated by hypoxia-inducible factor 1 (HIF-1), which is a heterodimeric transcription factor consisting ofαandβsubunits. HIF-1βis constantly expressed, whereas HIF-1αis degraded under normal oxygen conditions. Hypoxia stabilizes HIF-1αand the HIF complex, and HIF then translocates into the nucleus to initiate the expression of target genes. Hypoxia has been extensively studied for its role in promoting tumor progression, and emerging evidence also indicates that hypoxia may play important roles in physiological processes, including mammary development and lactation. The mammary gland exhibits an increasing metabolic rate from pregnancy to lactation to support mammary growth, lactogenesis, and lactation. This process requires increasing amounts of oxygen consumption and results in localized chronic hypoxia as confirmed by the binding of the hypoxia marker pimonidazole HCl in mouse mammary gland. We hypothesized that this hypoxic condition promotes mammary development and lactation, a hypothesis that is supported by the following several lines of evidence:i) Mice with an HIF-1αdeletion selective for the mammary gland have impaired mammary differentiation and lipid secretion, resulting in lactation failure and striking changes in milk compositions;ii) We recently observed that hypoxia significantly induces HIF-1α-dependent glucose uptake and GLUT1 expression in mammary epithelial cells, which may be responsible for the dramatic increases in glucose uptake and GLUT1 expression in the mammary gland during the transition period from late pregnancy to early lactation;and ii ) Hypoxia and HIF-1αincrease the

  16. Interrogation of the rat mammary gland using intraductal impedance spectroscopy

    International Nuclear Information System (INIS)

    Extant technologies for the detection of breast cancer exploit changes in the morphology of the mammary ductal epithelial network and can involve ionizing radiation. Intraductal surveillance of mammary epithelium has the potential to allow for earlier detection based on changes in function of the epithelium. This study investigated the feasibility of using intraductal impedance spectroscopy (IIS) to assess changes in resistance in the mammary epithelium in a small group of female rats in resting, pregnant and ultimately lactating states. In resting rats, intraductal surveillance was able to detect only a single resistive capacitance (RC). In pregnant animals, a second RC became evident in the frequency range between 1 and 190 Hz. The real resistance of this low frequency RC increased when measurements were made after the animals had begun lactating. Equivalent circuit modeling revealed this increase to be a 1.7-fold change from pregnancy to lactation. A model of tight junction closure in the context of ductal expansion is proposed. These results suggest that physiologic measurements can be made in rodent mammary epithelium using this technique allowing for assessment of function in normal and disease states

  17. Collagen density promotes mammary tumor initiation and progression

    Directory of Open Access Journals (Sweden)

    Knittel Justin G

    2008-04-01

    Full Text Available Abstract Background Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen. Thus, the influence of the extracellular matrix on breast carcinoma development and the underlying molecular mechanisms are not understood. Methods To study the effects of collagen density on mammary tumor formation and progression, we utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue. Imaging of the tumors and tumor-stromal interface in live tumor tissue was performed with multiphoton laser-scanning microscopy to generate multiphoton excitation and spectrally resolved fluorescent lifetimes of endogenous fluorophores. Second harmonic generation was utilized to image stromal collagen. Results Herein we demonstrate that increased stromal collagen in mouse mammary tissue significantly increases tumor formation approximately three-fold (p p Conclusion This study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. Furthermore, the imaging techniques and signature identified in this work may provide useful diagnostic tools to rapidly assess fresh tissue biopsies.

  18. Experimental manipulation of radiographic density in mouse mammary gland

    International Nuclear Information System (INIS)

    Extensive mammographic density in women is associated with increased risk for breast cancer. Mouse models provide a powerful approach to the study of human diseases, but there is currently no model that is suited to the study of mammographic density. We performed individual manipulations of the stromal, epithelial and matrix components of the mouse mammary gland and examined the alterations using in vivo and ex vivo radiology, whole mount staining and histology. Areas of density were generated that resembled densities in mammographic images of the human breast, and the nature of the imposed changes was confirmed at the cellular level. Furthermore, two genetic models, one deficient in epithelial structure (Pten conditional tissue specific knockout) and one with hyperplastic epithelium and mammary tumors (MMTV-PyMT), were used to examine radiographic density. Our data show the feasibility of altering and imaging mouse mammary gland radiographic density by experimental and genetic means, providing the first step toward modelling the biological processes that are responsible for mammographic density in the mouse

  19. Characterization and regulation of the bovine stearoyl-CoA desaturase gene promoter

    International Nuclear Information System (INIS)

    The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36 bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid

  20. Promoting effects of Chinese pangolin and wild pink medicines on the mammary gland development in immature mice.

    Science.gov (United States)

    Bayin, Jiragara; Matsumoto, Mitsuharu; Islam, Mohammad Saiful; Yabuki, Akira; Kanouchi, Hiroaki; Oka, Tatsuzo; Nishinakagawa, Hayao

    2009-10-01

    The effects of the mixture of crude aqueous extracts from Chinese pangolin and wild pink (C+P), traditional Chinese medicine, on the proliferation and differentiation of mammary gland epithelium in intact and ovariectomized immature mice were investigated by light and electron microscopy and BrdU immunohistochemistry. Although there were no significant differences in mammary gland fat pad and parenchyma areas between the intact experimental groups, the numbers of duct branchings and buds were significantly larger in the C+W treated mice than in the control mice. The ratio of BrdU immunopositive cells to total epithelial cells was higher in C+W treated intact mice. Ultrastructurally, epithelial cells of the mammary buds and ducts possessed an oval and lucent nucleus, and ribosomes increased in number or developed to a greater degree in C+W treated intact mice than in the control mice. Conversely, there were no significant differences in any measurements of mammary gland between the experimental groups of ovariectomized mice. BrdU immunoreactive cells were never seen and the ultrastructure of mammary epihelial cells indicated the inactive cell phase in both ovariectomied mice. In comparison between the intact and overiectomized mice, the mammary fat pad area was larger in the ovariectomized mice than in the intact mice, although another four measurements were larger in the intact groups. These observations suggest that administration with C+W could promote the development of mammary glands via ovary in immature mice. PMID:19887738

  1. Insufficiency of transformation by simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes for conversion of ethanolamine-responsive mammary cells to ethanolamine-nonresponsive cells.

    OpenAIRE

    Kano-Sueoka, T; King, D M

    1988-01-01

    Normal mammary epithelial cells (ethanolamine responsive) require ethanolamine to enable them to grow in defined culture medium because they cannot synthesize de novo a sufficient amount of phosphatidylethanolamine. Mammary tumor cells which retain properties of the normal tissue are also likely to be ethanolamine responsive, whereas dedifferentiated, highly tumorigenic mammary tumor cells are ethanolamine nonresponsive. The nonresponsive tumor cells are able to synthesize the necessary amoun...

  2. Intracellular behaviour of samarium and europium in lactating mammary gland

    Institute of Scientific and Technical Information of China (English)

    Ayadi Ahlem; Maghraoui Samira; El Hili Ali; Galle Pierre; Tekaya Leila

    2012-01-01

    The subcellular localization of samarium and europium,two rare-earths,increasingly used in both medical and industrial fields,has been studied in several organs such as liver and kidney but never in the mammary gland despite of its importance in the biology of lactation and nutrition domains.The intracellular behaviour of samarium and europium after their intra-peritoneal administration in the lactating mammary gland cells was investigated.The results showed the presence of very electron dense deposits in the glandular epithelial cell lysosomes.These particular lysosomes were never observed in the marnrnary cell lysosomes of control rats.These intralysosomal deposits were probably composed of insoluble samarium or europium phosphates by analogy with previous studies,the transmission electron microscopy,the ion mass microscopy and the electron probe microanalysis,and other techniques allowing the identification of the chemical structure of the intralysosomal deposits.

  3. Mammary tuberculosis: percutaneous treatment of a mammary tuberculous abscess

    International Nuclear Information System (INIS)

    It is currently very rare to find mammary involvement in cases of tuberculosis, in either primary or secondary form. Diagnosis is classically clinical and microbiological, and the basic techniques used in imaging diagnosis are mammography and ultrasound. Computed tomography may define the involvement of the thoracic wall in those cases which present as mammary masses adhering to deep levels, and is also able to evaluate accompanying pulmonary disease, if it is present. Traditionally, treatment has consisted of quadrantectomy and specific antibiotic therapy. We present a case of tuberculous mammary abscess secondary to pulmonary disease, which was treated by percutaneous drainage controlled by CT and specific antibiotic therapy. We revise the diagnosis, differential diagnosis and treatment of mammary tuberculosis. (orig.)

  4. Mammary tuberculosis: percutaneous treatment of a mammary tuberculous abscess

    Energy Technology Data Exchange (ETDEWEB)

    Romero, C.; Carreira, C.; Cereceda, C.; Pinto, J. [Servicio de Radiologia, Hospital Virgen de la Salud, Toledo (Spain); Lopez, R.; Bolanos, F. [Servicio de Cirugia, Hospital Virgen de la Salud, Toledo (Spain)

    2000-03-01

    It is currently very rare to find mammary involvement in cases of tuberculosis, in either primary or secondary form. Diagnosis is classically clinical and microbiological, and the basic techniques used in imaging diagnosis are mammography and ultrasound. Computed tomography may define the involvement of the thoracic wall in those cases which present as mammary masses adhering to deep levels, and is also able to evaluate accompanying pulmonary disease, if it is present. Traditionally, treatment has consisted of quadrantectomy and specific antibiotic therapy. We present a case of tuberculous mammary abscess secondary to pulmonary disease, which was treated by percutaneous drainage controlled by CT and specific antibiotic therapy. We revise the diagnosis, differential diagnosis and treatment of mammary tuberculosis. (orig.)

  5. Cdk2-Null Mice Are Resistant to ErbB-2-Induced Mammary Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Dipankar Ray

    2011-05-01

    Full Text Available The concept of targeting G1 cyclin-dependent kinases (CDKs in breast cancer treatments is supported by the fact that the genetic ablation of Cdk4 had minimal impacts on normal cell proliferation in majority of cell types, resulting in near-normal mouse development, whereas such loss of Cdk4 completely abrogated ErbB-2/neu-induced mammary tumorigenesis in mice. In most human breast cancer tissues, another G1-regulatory CDK, CDK2, is also hyperactivated by various mechanisms and is believed to be an important therapeutic target. In this report, we provide genetic evidence that CDK2 is essential for proliferation and oncogenesis of murine mammary epithelial cells. We observed that 87% of Cdk2-null mice were protected from ErbB-2-induced mammary tumorigenesis. Mouse embryonic fibroblasts isolated from Cdk2-null mouse showed resistance to various oncogene-induced transformation. Previously, we have reported that hemizygous loss of Cdc25A, the major activator of CDK2, can also protect mice from ErbB-2-induced mammary tumorigenesis [Cancer Res (2007 67(14: 6605–11]. Thus, we propose that CDC25A-CDK2 pathway is critical for the oncogenic action of ErbB-2 in mammary epithelial cells, in a manner similar to Cyclin D1/CDK4 pathway.

  6. Global Changes in the Mammary Epigenome Are Induced by Hormonal Cues and Coordinated by Ezh2

    Directory of Open Access Journals (Sweden)

    Bhupinder Pal

    2013-02-01

    Full Text Available The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.

  7. Overexpression of miR-30b in the developing mouse mammary gland causes a lactation defect and delays involution.

    Directory of Open Access Journals (Sweden)

    Sandrine Le Guillou

    Full Text Available BACKGROUND: MicroRNA (miRNA are negative regulators of gene expression, capable of exerting pronounced influences upon the translation and stability of mRNA. They are potential regulators of normal mammary gland development and of the maintenance of mammary epithelial progenitor cells. This study was undertaken to determine the role of miR-30b on the establishment of a functional mouse mammary gland. miR-30b is a member of the miR-30 family, composed of 6 miRNA that are highly conserved in vertebrates. It has been suggested to play a role in the differentiation of several cell types. METHODOLOGY/PRINCIPAL FINDINGS: The expression of miR-30b was found to be regulated during mammary gland development. Transgenic mice overexpressing miR-30b in mammary epithelial cells were used to investigate its role. During lactation, mammary histological analysis of the transgenic mice showed a reduction in the size of alveolar lumen, a defect of the lipid droplets and a growth defect of pups fed by transgenic females. Moreover some mammary epithelial differentiated structures persisted during involution, suggesting a delay in the process. The genes whose expression was affected by the overexpression of miR-30b were characterized by microarray analysis. CONCLUSION/SIGNIFICANCE: Our data suggests that miR-30b is important for the biology of the mammary gland and demonstrates that the deregulation of only one miRNA could affect lactation and involution.

  8. Amino acids and mammary gland development: nutritional implications for milk production and neonatal growth.

    Science.gov (United States)

    Rezaei, Reza; Wu, Zhenlong; Hou, Yongqing; Bazer, Fuller W; Wu, Guoyao

    2016-01-01

    Milk is synthesized by mammary epithelial cells of lactating mammals. The synthetic capacity of the mammary gland depends largely on the number and efficiency of functional mammary epithelial cells. Structural development of the mammary gland occurs during fetal growth, prepubertal and post-pubertal periods, pregnancy, and lactation under the control of various hormones (particularly estrogen, growth hormone, insulin-like growth factor-I, progesterone, placental lactogen, and prolactin) in a species- and stage-dependent manner. Milk is essential for the growth, development, and health of neonates. Amino acids (AA), present in both free and peptide-bound forms, are the most abundant organic nutrients in the milk of farm animals. Uptake of AA from the arterial blood of the lactating dam is the ultimate source of proteins (primarily β-casein and α-lactalbumin) and bioactive nitrogenous metabolites in milk. Results of recent studies indicate extensive catabolism of branched-chain AA (leucine, isoleucine and valine) and arginine to synthesize glutamate, glutamine, alanine, aspartate, asparagine, proline, and polyamines. The formation of polypeptides from AA is regulated not only by hormones (e.g., prolactin, insulin and glucocorticoids) and the rate of blood flow across the lactating mammary gland, but also by concentrations of AA, lipids, glucose, vitamins and minerals in the maternal plasma, as well as the activation of the mechanistic (mammalian) target rapamycin signaling by certain AA (e.g., arginine, branched-chain AA, and glutamine). Knowledge of AA utilization (including metabolism) by mammary epithelial cells will enhance our fundamental understanding of lactation biology and has important implications for improving the efficiency of livestock production worldwide. PMID:27042295

  9. Mammary cells with active Wnt signaling resist ErbB2-induced tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Wen Bu

    Full Text Available Aberrant activation of Wnt signaling is frequent in human malignancies. In normal epithelial tissues, including the breast, Wnt signaling is active only in a subset of cells, but it is unknown whether this subset of Wnt signaling-active cells is at increased risk of carcinogenesis. We created transgenic mice (TOP-tva in which the synthetic Wnt-responsive promoter TOP controlled the gene encoding TVA, which confers susceptibility to infection by the retroviral vector RCAS. Thus, only cells in which Wnt signaling is active will express tva and be targeted by RCAS. Surprisingly, we found that RCAS-mediated delivery of cDNA encoding a constitutively activated version of ErbB2 (HER2/Neu into the small number of TVA+ mammary epithelial cells in TOP-tva mice failed to induce tumor, while the same virus readily induced mammary tumors after it was delivered into a comparable number of cells in our previously reported mouse line MMTV-tva, whose tva is broadly expressed in mammary epithelium. Furthermore, we could not even detect any early lesions or infected cells in TOP-tva mice at the time of necropsy. Therefore, we conclude that the Wnt pathway-active cell subset in the normal mammary epithelium does not evolve into tumors following ErbB2 activation-rather, they apparently die due to apoptosis, an anticancer "barrier" that we have reported to be erected in some mammary cells followed ErbB2 activation. In accord with these mouse model data, we found that unlike the basal subtype, ErbB2+ human breast cancers rarely involve aberrant activation of Wnt signaling. This is the first report of a defined sub-population of mammalian cells that is "protected" from tumorigenesis by a potent oncogene, and provides direct in vivo evidence that mammary epithelial cells are not equal in their response to oncogene-initiated transformation.

  10. The Expression of the IGF Family During Mouse Mammary Gland Development

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study was to determine the patterns and levels of IGF family members' expression during postnatal mammary gland development. The authors investigated the protein expression profile of the major components of the IGF axis in murine mammary glands. All the proteins examined, IGF- Ⅰ, IGF- Ⅱ, and IGF- Ⅰ receptor (IGF- Ⅰ R) were expressed at greatly different levels and displayed unique expression profiles. IGF- Ⅱ and IGF- Ⅰ R were always expressed at significantly higher levels than IGF- Ⅰ. IGF- Ⅰ was localized in adipocytes as well as the epithelial and stromal compartments, but just distinctly expressed where mammary cells aggregated to form ducts, in virgins. The IGF- Ⅱ was localized only on the basal layer epithelial cell membranes of ducts and alveoli, with a peak level on the initiation of lactation. The higher level of IGF- Ⅰ R compared with IGF- Ⅰ was also found in adipocytes as well as in the epithelial and stromal compartments, especially during pregnancy and late lactation. The IGF- Ⅰ R pathway was obviously significant for the development of the mammary parenchyma and stroma. Overall, the comparison of the expression profiles of these different proteins would strongly suggest that they were likely to have different functions throughout the mammary gland development, and it also highlighted the potential interactions and coregulation of the members of this axis. It seems that IGF- Ⅱ was the major local modulator rather than IGF- Ⅰ by an IGF- Ⅰ R-independent pathway, especially for initiation of lactation. This study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.

  11. Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Pujuguet, Philippe; Simian, Marina; Liaw, Jane; Timpl, Rupert; Werb, Zena; Bissell, Mina J..

    2000-02-01

    Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express b-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone to mammary epithelial cells induced b- casein production. We asked whether recombinant nidogen-1 alone could signal directly for b-casein. Nidogen-1 did not induce b-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate b-casein expression. Addition of full length nidogen-1 to the mixed cultures had no effect on b-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on b-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.

  12. Biological and genetic properties of the p53 null preneoplastic mammary epithelium

    Science.gov (United States)

    Medina, Daniel; Kittrell, Frances S.; Shepard, Anne; Stephens, L. Clifton; Jiang, Cheng; Lu, Junxuan; Allred, D. Craig; McCarthy, Maureen; Ullrich, Robert L.

    2002-01-01

    The absence of the tumor suppressor gene p53 confers an increased tumorigenic risk for mammary epithelial cells. In this report, we describe the biological and genetic properties of the p53 null preneoplastic mouse mammary epithelium in a p53 wild-type environment. Mammary epithelium from p53 null mice was transplanted serially into the cleared mammary fat pads of p53 wild-type BALB/c female to develop stable outgrowth lines. The outgrowth lines were transplanted for 10 generations. The outgrowths were ductal in morphology and progressed through ductal hyperplasia and ductal carcinoma in situ before invasive cancer. The preneoplastic outgrowth lines were immortal and exhibited activated telomerase activity. They are estrogen and progesterone receptor-positive, and aneuploid, and had various levels of tumorigenic potential. The biological and genetic properties of these lines are distinct from those found in most hyperplastic alveolar outgrowth lines, the form of mammary preneoplasia occurring in most traditional models of murine mammary tumorigenesis. These results indicate that the preneoplastic cell populations found in this genetically engineered model are similar in biological properties to a subset of precurser lesions found in human breast cancer and provide a unique model to identify secondary events critical for tumorigenicity and invasiveness.

  13. Canine mammary tumors - clinical survey

    Directory of Open Access Journals (Sweden)

    Elena Atanaskova Petrov

    2014-10-01

    Full Text Available Mammary tumours are the second most frequent neoplasia in dogs, mainly affecting older female patients. Approximately 50% of the mammary tumours are malignant with high percentage of mortality if not treated in time. The aim of this study was to analyze the data of canine patients with mammary tumours, to evaluate the type of tumours, as well as the relationship between tumour incidence and dogs’ age, reproductive cycle and sterilization. The survey was used to retrieve the information in the period of two years from the patient data base of the University Veterinary Hospital at the Faculty of Veterinary medicine in Skopje. Patients included in this survey were subjected to routine clinical investigation and additional laboratory tests (cytological examination, x-rays imaging, CBC and biochemical profile, histopathology of the tumor samples. Aged female patients (12 – 13 years are the most susceptible category for development of mammary tumours. The reproductive history showed that five of the patients with malignant mammary tumourshave never whelped and were not treated with any exogenous hormones. Malignant tumours (adenocarcinoma were diagnosed in 90% of the patients. Three patients died due to lung metastasis. Late diagnosis is one of the major problems that results in lethal outcome due to lung metastases. Since ovarian steroids play an important role in the aetiology, the most effective prevention of mammary tumoursis elective ovariectomy of the bitch at an early age.

  14. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells

    International Nuclear Information System (INIS)

    Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors. In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists. Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells. These studies

  15. Chemoprevention of Radiation Induced Rat Mammary Neoplasms

    Science.gov (United States)

    Huso, David L.

    1999-01-01

    Radiations encountered in space include protons and heavy ions such as iron as well as their secondaries. The relative biological effect (RBE) of these ions is not known, particularly at the doses and dose-rates expected for planetary missions. Neutrons, are not particularly relevant to space travel, but have been found experimentally to have an increase in their RBE with decreasing dose. If a similar trend of increasing RBE with decreasing dose is present for heavy ions and protons during irradiation in space, the small doses received during space travel could potentially have substantial carcinogenic risk. Clearly more investigation of the effects of heavy ions and protons is needed before accurate risk assessment for prolonged travel in space can be done. One means to mitigate the increased risk of cancer due to radiation exposure in space is by developing effective countermeasures that can reduce the incidence of tumor development. Tamoxifen has recently been shown to be an effective chemopreventive agent in both animal models and humans for the prevention of mammary tumors. Tamoxifen is a unique drug, with a highly specific mechanism of action affecting a specific radiation-sensitive population of epithelial cells in the mammary gland. In human studies, the annual incidence of a primary tumor in the contralateral breast of women with previous breast cancer is about 8 per 1000, making them an exceedingly high-risk group for the development of breast cancer. In this high risk group, treated with tamoxifen, daily, for 2 years, the incidence of a new primary tumor in the contralateral breast was approximately one third of that noted in the non-tamoxifen treatment group. Tamoxifen antagonizes the action of estrogen by competing for the nuclear receptor complex thereby altering the association of the receptor complex and nuclear binding sites. Its effects in reducing the development of breast cancer could be accomplished by controlling clinically undetectable

  16. The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage.

    Directory of Open Access Journals (Sweden)

    Nisha M Badders

    Full Text Available BACKGROUND: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%. Stem cell activity can be enriched by >200 fold (over 80% of activity, based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22% and Lrp5-/- mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16(Ink4a and TA-p63. CONCLUSIONS/SIGNIFICANCE: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.

  17. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide

    OpenAIRE

    Vels Lotte; Røntved Christine; Sørensen Peter; Jiang Li; Ingvartsen Klaus L

    2008-01-01

    Abstract Background Liver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM) exposure to E. coli lipopolysaccharide (LPS) treatment. Results Approximately 20% target transcripts were d...

  18. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide

    OpenAIRE

    Jiang, Li; Sørensen, Peter; Røntved, Christine; Vels, Lotte; Ingvartsen, Klaus L

    2008-01-01

    Background Liver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM) exposure to E. coli lipopolysaccharide (LPS) treatment. Results Approximately 20% target transcripts were differenti...

  19. Single Colony Preparation and Amplification Culture of Fetal Fibroblast and Mammary Gland Epithelial Cell of Human Lactoferrin Transgene from Goat%山羊转人乳铁蛋白基因成纤维细胞和乳腺上皮细胞单克隆的制备及扩大培养

    Institute of Scientific and Technical Information of China (English)

    张玉玲; 刘凤军; 张涌

    2009-01-01

    [ Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C, which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [ Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells. Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture ( FF: 53.33% vs. 10.00% ;MGE; 33. 33% vs. 6. 67% ) , confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [ Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.%[目的]为探索单个转基因阳性细胞快速扩大培养成为细胞克隆的技术体系.[方法]对转染人乳铁蛋白(Human lactoferrin,hLF)基因乳腺特异性表达载体pBLM-C1的单个山羊胎儿成纤维细胞(Fetal Fibroblasts,FF)和乳腺上皮细胞(Mammary Gland Epithelial,MGE)细胞进行克隆.在96孔板中,首先用3种浓度(V/V: 0 、50%和100%)的适应性条件培养基对单个转染细胞进行细胞单克隆的制备,进而把转染细胞单克隆与非转

  20. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  1. Species distribution and resistance profiles of coagulase-negative staphylococci isolated from bovine mastitis in Switzerland

    OpenAIRE

    Moser, A.; Stephan, R.; DE ZIEGLER, D.; Johler, S.

    2013-01-01

    Coagulase-negative staphylococci (CNS) are the predominant cause of bovine intra-mammary infections. They can lead to chronic infections and were reported to significantly increase milk somatic cell counts. The goal of our study was to determine the species distribution and antimicrobial susceptibility profiles of CNS in bovine mastitis milk samples in Switzerland. Between March 2011 and February 2012, a total of 120 CNS were isolated from mastitis milk samples from 117 different animals at 7...

  2. Population structure and antimicrobial profile of Staphylococcus aureus strains associated with bovine mastitis in China.

    Science.gov (United States)

    Zhang, Lili; Li, Yuchen; Bao, Hongduo; Wei, Ruicheng; Zhou, Yan; Zhang, Hui; Wang, Ran

    2016-08-01

    Staphylococcus aureus is a significant bacterial pathogen associated with bovine mastitis. The aim of the present study was to investigate and characterize of S. aureus strains isolated from the milk of cows suffering from mastitis in the mid-east of China. Among the 200 milk samples analyzed, 58 were positive for S. aureus, of these isolates, 11 isolates were methicillin-resistant Staphylococcus aureus (MRSA). All of the 58 S. aureus strains were classified in agr group I, while seven different sequence type (ST) patterns were identified and among them the most common was ST630 followed by ST188. All of the S. aureus isolates belonging to ST630 were resistant to more than four antimicrobials, and 22.2% of isolates belonging to ST188 were resistant to eight antimicrobials. Interestingly, while strong biofilm producers demonstrated higher resistance to multiple antimicrobials, they exhibited lower intracellular survival rates. The results of this study illustrated the distribution, antimicrobial susceptibility profiles, genotype, and the ability of biofilm production and mammary epithelial cells invasion of these S. aureus isolates. This study can provide the basis for the development of a disease prevention program in dairy farms to reduce the potential risk in both animal and human health. PMID:27265679

  3. Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia

    Science.gov (United States)

    Holst, Charles R.; Nuovo, Gerard J.; Esteller, Manel; Chew, Karen; Baylin, Stephen B.; Herman, James G.; Tlsty, Thea D.

    2003-01-01

    Cultures of human mammary epithelial cells (HMECs) contain a subpopulation of variant cells with the capacity to propagate beyond an in vitro proliferation barrier. These variant HMECs, which contain hypermethylated and silenced p16(INK4a) (p16) promoters, eventually accumulate multiple chromosomal changes, many of which are similar to those detected in premalignant and malignant lesions of breast cancer. To determine the origin of these variant HMECs in culture, we used Luria-Delbruck fluctuation analysis and found that variant HMECs exist within the population before the proliferation barrier, thereby raising the possibility that variant HMECs exist in vivo before cultivation. To test this hypothesis, we examined mammary tissue from normal women for evidence of p16 promoter hypermethylation. Here we show that epithelial cells with methylation of p16 promoter sequences occur in focal patches of histologically normal mammary tissue of a substantial fraction of healthy, cancer-free women.

  4. Mammary blood flow and nutrient uptake

    OpenAIRE

    Farmer, Chantal; Trottier, N.L.; Dourmad, Jean-Yves

    2015-01-01

    Sow milk is the major source of nutrients for suckling piglets and taking into account the large litter sizes of our current sow genotypes, it is imperative to maximize nutrient use by the mammary gland. The amount of nutrients available to mammary tissue is dependent upon the concentrations of nutrients in blood and the rate of its flow to the lactating glands. Nutrient availability to the udder may be estimated by measuring mammary arteriovenous differences, and mammary blood flow can be me...

  5. Mammary carcinoma diagnostics and therapy; Diagnostik und Therapie des Mammakarzinoms

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Uwe; Baum, Friedemann (eds.) [Diagnostisches Brustzentrum Goettingen BZG, Goettingen(Germany)

    2014-11-01

    The book on mammary carcinoma diagnostics and therapy covers the following issues: development, anatomy and physiology of the mammary glands, pathology of benign and malign mammary gland changes, non-imaging diagnostics; mammography; ultrasonic mammography; magnetic resonance tomography of the mammary glands; imaging diagnostics findings; mammary interventions; examination concepts; operative therapy of the mammary carcinoma; chemotherapy of the mammary carcinoma; radio-oncological therapy of the mammary carcinoma; logistics in a medical center for mammary gland diseases; logistics in an interdisciplinary center for mammary diseases; dialogue conduction and psycho-social attendance.

  6. Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for transcription of the gene encoding beta-casein.

    OpenAIRE

    Schmitt-Ney, M; Happ, B; Ball, R K; Groner, B

    1992-01-01

    During the lactation period, mammary epithelial cells secrete large amounts of milk proteins. The coordinate regulation of milk protein expression is effected by the lactogenic hormones. We have investigated the activity of a mammary gland-specific transcription factor (MGF), which mediates hormonal influences at the level of a milk protein gene promoter. MGF-binding sites are present in the promoters of the most abundantly expressed milk protein genes. Mutation of the MGF-binding site in the...

  7. Akt1 is essential for postnatal mammary gland development, function, and the expression of Btn1a1.

    Directory of Open Access Journals (Sweden)

    Jessica LaRocca

    Full Text Available Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt1-/- C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt1-/- mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1-/- mammary glands. Additionally, pseudopregnant Akt1-/- females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function.

  8. Methods in Mammary Gland Development and Cancer: the second ENDBC meeting - intravital imaging, genomics, modeling and metastasis

    OpenAIRE

    Stingl, John; Matthew J Smalley; Glukhova, Marina A.; Bentires-Alj, Mohamed

    2010-01-01

    The second meeting of the European Network for Breast Development and Cancer (ENBDC) on 'Methods in Mammary Gland Development and Cancer' was held in April 2010 in Weggis, Switzerland. The focus was on genomics and bioinformatics, extracellular matrix and stroma-epithelial cell interactions, intravital imaging, the search for metastasis founder cells and mouse models of breast cancer.

  9. Zinc Finger Homeodomain Factor Zfhx3 Is Essential for Mammary Lactogenic Differentiation by Maintaining Prolactin Signaling Activity.

    Science.gov (United States)

    Zhao, Dan; Ma, Gui; Zhang, Xiaolin; He, Yuan; Li, Mei; Han, Xueying; Fu, Liya; Dong, Xue-Yuan; Nagy, Tamas; Zhao, Qiang; Fu, Li; Dong, Jin-Tang

    2016-06-10

    The zinc finger homeobox 3 (ZFHX3, also named ATBF1 for AT motif binding factor 1) is a transcription factor that suppresses prostatic carcinogenesis and induces neuronal differentiation. It also interacts with estrogen receptor α to inhibit cell proliferation and regulate pubertal mammary gland development in mice. In the present study, we examined whether and how Zfhx3 regulates lactogenic differentiation in mouse mammary glands. At different stages of mammary gland development, Zfhx3 protein was expressed at varying levels, with the highest level at lactation. In the HC11 mouse mammary epithelial cell line, an in vitro model of lactogenesis, knockdown of Zfhx3 attenuated prolactin-induced β-casein expression and morphological changes, indicators of lactogenic differentiation. In mouse mammary tissue, knock-out of Zfhx3 interrupted lactogenesis, resulting in underdeveloped glands with much smaller and fewer alveoli, reduced β-casein expression, accumulation of large cytoplasmic lipid droplets in luminal cells after parturition, and failure in lactation. Mechanistically, Zfhx3 maintained the expression of Prlr (prolactin receptor) and Prlr-Jak2-Stat5 signaling activity, whereas knockdown and knock-out of Zfhx3 in HC11 cells and mammary tissues, respectively, decreased Prlr expression, Stat5 phosphorylation, and the expression of Prlr-Jak2-Stat5 target genes. These findings indicate that Zfhx3 plays an essential role in proper lactogenic development in mammary glands, at least in part by maintaining Prlr expression and Prlr-Jak2-Stat5 signaling activity. PMID:27129249

  10. Differentiation-induced cleavage of Cutl1/CDP generates a novel dominant-negative isoform that regulates mammary gene expression.

    Science.gov (United States)

    Maitra, Urmila; Seo, Jin; Lozano, Mary M; Dudley, Jaquelin P

    2006-10-01

    Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland. PMID:17015474

  11. Mammary carcinosarcoma in cat: a case report

    OpenAIRE

    J.D.G. Paniago; A.L.S. Vieira; N.M. Ocarino; S.A. França; C. Malm; Cassali, G.D.; R. Serakides

    2010-01-01

    A case of mammary carcinosarcoma is reported in a 13-year-old, mixed breed female cat, which was not spayed and had not received contraceptives. The patient presented extensive and coalescent nodules in all mammary glands. Based on the histological and immunohistochemical findings, the diagnosis of mammary carcinosarcoma was confirmed.

  12. Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

    Science.gov (United States)

    Blacher, Silvia; Gérard, Céline; Gallez, Anne; Foidart, Jean-Michel; Noël, Agnès; Péqueux, Christel

    2016-04-01

    The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology. PMID:26910307

  13. Autocrine-paracrine regulation of the mammary gland.

    Science.gov (United States)

    Weaver, S R; Hernandez, L L

    2016-01-01

    The mammary gland has a remarkable capacity for regulation at a local level, particularly with respect to its main function: milk secretion. Regulation of milk synthesis has significant effects on animal and human health, at the level of both the mother and the neonate. Control by the mammary gland of its essential function, milk synthesis, is an evolutionary necessity and is therefore tightly regulated at a local level. For at least the last 60 yr, researchers have been interested in elucidating the mechanisms underpinning the mammary gland's ability to self-regulate, largely without the influence from systemic hormones or signals. By the 1960s, scientists realized the importance of milk removal in the capacity of the gland to produce milk and that the dynamics of this removal, including emptying of the alveolar spaces and frequency of milking, were controlled locally as opposed to traditional systemic hormonal regulation. Using both in vitro systems and various mammalian species, including goats, marsupials, humans, and dairy cows, it has been demonstrated that the mammary gland is largely self-regulating in its capacity to support the young, which is the evolutionary basis for milk production. Local control occurs at the level of the mammary epithelial cell through pressure and stretching negative-feedback mechanisms, and also in an autocrine fashion through bioactive factors within the milk which act as inhibitors, regulating milk secretion within the alveoli themselves. It is only within the last 20 to 30 yr that potential candidates for these bioactive factors have been examined at a molecular level. Several, including parathyroid hormone-related protein, growth factors (transforming growth factor, insulin-like growth factor, epidermal growth factor), and serotonin, are synthesized within and act upon the gland and possess dynamic receptor activity resulting in diverse effects on growth, calcium homeostasis, and milk composition. This review will focus on the

  14. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

    Directory of Open Access Journals (Sweden)

    Kamini Kunasegaran

    Full Text Available The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production. Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

  15. Histological analysis of low dose NMU effects in the rat mammary gland

    Directory of Open Access Journals (Sweden)

    Sonnenschein Carlos

    2009-08-01

    Full Text Available Abstract Background Our objective was to assess the histological changes in mammary glands of the female Wistar-Furth rat as a result of low dose exposure to N-nitrosomethylurea (NMU. Methods Groups of 30–40 virgin female rats of between 49–58 days old received a single injection of 10, 20, 30 or 50 mg NMU/kg body weight (BW. A group of 10 control rats received 0.9% NaCl solution only. The formation of palpable mammary gland tumors was assessed weekly and, upon sacrifice at 12, 22 and 25–30 weeks after treatment, we performed a comprehensive histological analysis of all mammary gland lesions and tumors. Results Alongside the predicted increase in tumor number and decrease in tumor latency with increasing NMU dose, we observed a number of microscopic lesions and other epithelial abnormalities in the mammary glands for all NMU doses. Two types of non-neoplastic histological changes were observed in rats exposed to 10 or 20 mg NMU/kg BW: namely, (i an increase in the number of acinar structures often accompanied by secretion into the lumen which is normally associated with pregnancy and lactation, and (ii an increase in the number of epithelial cells sloughed into the lumen of the epithelial ducts. Conclusion This study establishes a baseline for low-dose exposure and defines the histological features in the mammary gland resulting from NMU exposure. Furthermore, this system provides an ideal platform for evaluating the relative susceptibility of animals protected from, or predisposed to, developing cancer through environmental influences.

  16. Histological analysis of low dose NMU effects in the rat mammary gland

    International Nuclear Information System (INIS)

    Our objective was to assess the histological changes in mammary glands of the female Wistar-Furth rat as a result of low dose exposure to N-nitrosomethylurea (NMU). Groups of 30–40 virgin female rats of between 49–58 days old received a single injection of 10, 20, 30 or 50 mg NMU/kg body weight (BW). A group of 10 control rats received 0.9% NaCl solution only. The formation of palpable mammary gland tumors was assessed weekly and, upon sacrifice at 12, 22 and 25–30 weeks after treatment, we performed a comprehensive histological analysis of all mammary gland lesions and tumors. Alongside the predicted increase in tumor number and decrease in tumor latency with increasing NMU dose, we observed a number of microscopic lesions and other epithelial abnormalities in the mammary glands for all NMU doses. Two types of non-neoplastic histological changes were observed in rats exposed to 10 or 20 mg NMU/kg BW: namely, (i) an increase in the number of acinar structures often accompanied by secretion into the lumen which is normally associated with pregnancy and lactation, and (ii) an increase in the number of epithelial cells sloughed into the lumen of the epithelial ducts. This study establishes a baseline for low-dose exposure and defines the histological features in the mammary gland resulting from NMU exposure. Furthermore, this system provides an ideal platform for evaluating the relative susceptibility of animals protected from, or predisposed to, developing cancer through environmental influences

  17. Intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: first clinical application.

    Science.gov (United States)

    Lange-Consiglio, A; Spelta, C; Garlappi, R; Luini, M; Cremonesi, F

    2014-10-01

    Bovine udder infections induce a variety of changes in gene expression of different growth factors that may suggest their possible role in glandular tissue protection or repair processes. Growth factors and also chemokines and cytokines may act synergistically to increase the infiltration of neutrophils and macrophages to promote angiogenesis, fibroplasia, matrix deposition, and, ultimately, re-epithelialization. Considering the vast applications, typically in human medicine, of platelet concentrate (PC) and its ease of preparation, the aim of our study was to evaluate an alternative therapy to stimulate the regeneration of glandular tissue, administering a concentration in excess of the growth factors contained in the PC. In each one of the 3 farms examined in the trial, PC was prepared from donor cows in good health, free from infections, and with no records of medications administered during the previous 2 mo. The platelet produced in one farm was used only for treating the cows of the same farm in a heterologous way. A total of 229 mastitic quarters were divided in 3 groups: antibiotic group (treated with intramammary antibiotic), antibiotic and PC group (treated intramammarily with antibiotics in association with PC), and PC group (treated with intramammary PC alone). The diagnosis of mastitis was based on somatic cell count and bacteriological evaluation of the milk from the affected quarter. Platelet concentrate, alone or in association with antibiotic, was used for 3 consecutive days as an unconventional therapy in bovine acute and chronic mastitis. Our data show that the associated action of antibiotic and PC performed significantly better than the antibiotic alone, either for the recovery of the affected mammary quarters or for somatic cell count reduction. In the same way, the association antibiotic plus PC showed significantly fewer relapses compared with the antibiotic alone, either for acute or chronic mastitis. The treatment with only PC did not show

  18. Morphological changes induced by testosterone in the mammary glands of female Wistar rats

    Directory of Open Access Journals (Sweden)

    A. Chambô-Filho

    2005-04-01

    Full Text Available Increased levels of androgens in postmenopausal women are considered to be a risk factor for breast cancer. Testosterone, alone or in combination with estrogen, induces epithelial dysplasia and mammary tumors in Noble rats. Since this model of hormone-induced neoplasia has not been reported in other rat strains, we studied the effect of testosterone on the mammary gland morphology of female Wistar rats. Sixty adult, non-castrated, female Wistar rats were implanted in the dorsum midline with a silicone tube containing 50 mg testosterone (testosterone propionate in 30 animals and non-esterified testosterone in the remaining 30 animals and 20 additional animals were implanted with empty tubes and used as control. Five animals per group were killed 30, 60, 90, 120, 150, and 180 days after implantation, and the mammary glands were dissected, fixed and embedded in paraffin. Histological sections were then stained with hematoxylin and eosin and picrosyrius red for collagen visualization. Morphological and morphometric analysis demonstrated ductal proliferation and acinotubular differentiation with secretory activity in all treated animals, peaking at 90 days of androgen exposure. After 90 days the proliferation of acinar epithelial cells was evident, but there was a progressive reduction of secretory differentiation and an increase in intralobular collagen fibers. There was no morphological evidence of dysplastic changes or other pre-neoplastic lesions. Testosterone treatment applied to adult, non-castrated female Wistar rats induced a mammary gland hyperplasia resembling the lactating differentiation, with progressive reduction in secretory differentiation.

  19. Genome Sequence Analysis of Staphylococcus equorum Bovine Mastitis Isolate UMC-CNS-924

    OpenAIRE

    Calcutt, Michael J.; Foecking, Mark F.; Hsieh, Hsin-Yeh; Perry, Jeanette; Stewart, George C.; Middleton, John R.

    2013-01-01

    Intramammary infections in dairy cattle are frequently caused by staphylococci, resulting in mastitis and associated economic losses. A draft genome sequence was determined for Staphylococcus equorum UMC-CNS-924, isolated from the milk of a Holstein cow, to better understand the genetic basis of its pathogenesis and adaptation to the bovine mammary gland.

  20. Histological and Morphometric Study of Regressive Changes in the Mammary Glands of Pigs during Regression

    Directory of Open Access Journals (Sweden)

    Svätoslav Hluchý

    2011-10-01

    Full Text Available The work deals with the histological and morphometric description of regressive changes in the mammary glands of swine after lactation. Samples of mammary glands of thoracic, abdominal and pubic udders were taken soon after they were killed. Samples were processed by histological and histochemical methods and evaluated by subjective and quantitative morphometrical methods. It was found that post-lactation increases the relative volume of connective stroma up to 75.10 ± 14.76 % of collagen in the tissue falls to 74.13 ± 14.87 % and the loose connective tissue, which was part of glandular parenchyma lobules accounts for only 0.97 ± 2.13%. Adipose tissue is 18.93 ± 14.93% of the udders volume.. Relative of glandular parenchyma fell to 5.97 ± 4.95% and mainly consists of a interlobular and lobar ducts and atrophic alveoli. Epithelium is generally in the mammary glands 4.22 ± 3.56% and lumen 1.75 ±1.81%. 1 cm3 of tissue in the mammary glands located 190745.52 tubuloalveolar structures. Alveoli reach an average size of 36.80 ± 11.57 μm. It was found relative volume of the walls (epithelium and cavities (lumen of each alveolus, the amount of epithelial cells and the nucleocytoplasmic ratio. The individual building components mammary glands were calculated correlation relations.

  1. Janus Kinase 1 Is Essential for Inflammatory Cytokine Signaling and Mammary Gland Remodeling.

    Science.gov (United States)

    Sakamoto, Kazuhito; Wehde, Barbara L; Yoo, Kyung Hyun; Kim, Taemook; Rajbhandari, Nirakar; Shin, Ha Youn; Triplett, Aleata A; Rädler, Patrick D; Schuler, Fabian; Villunger, Andreas; Kang, Keunsoo; Hennighausen, Lothar; Wagner, Kay-Uwe

    2016-06-01

    Despite a wealth of knowledge about the significance of individual signal transducers and activators of transcription (STATs), essential functions of their upstream Janus kinases (JAKs) during postnatal development are less well defined. Using a novel mammary gland-specific JAK1 knockout model, we demonstrate here that this tyrosine kinase is essential for the activation of STAT1, STAT3, and STAT6 in the mammary epithelium. The loss of JAK1 uncouples interleukin-6-class ligands from their downstream effector, STAT3, which leads to the decreased expression of STAT3 target genes that are associated with the acute-phase response, inflammation, and wound healing. Consequently, JAK1-deficient mice exhibit impaired apoptosis and a significant delay in mammary gland remodeling. Using RNA sequencing, we identified several new JAK1 target genes that are upregulated during involution. These include Bmf and Bim, which are known regulators of programmed cell death. Using a BMF/BIM-double-knockout epithelial transplant model, we further validated that the synergistic action of these proapoptotic JAK1 targets is obligatory for the remodeling of the mammary epithelium. The collective results of this study suggest that JAK1 has nonredundant roles in the activation of particular STAT proteins and this tyrosine kinase is essential for coupling inflammatory cytokine signals to the cell death machinery in the differentiated mammary epithelium. PMID:27044867

  2. The Evolution of mammary glands at different stages in Sarda dairy ewes: preliminary results

    OpenAIRE

    Anna Nudda; Natalia Castañares Castro; Alessandro Mazzette; Giuliana Canu; Maura Farinacci; Giuseppe Pulina; Monica Colitti

    2009-01-01

    The significance of cytological evolution of alveolar cells explains differences in milk yield that resulted in a different shape of lactation curve. In this paper, investigation of cellular background for this pattern was studied morphometrically in mammary gland of dairy ewes prior to lambing to involution. The ratio of epithelial to the luminal area was greatest at 7 days from lambing, it diminished on day 30 and 60 of lactation and it increased thereafter on day 150 of lactation and on da...

  3. Accelerated apoptosis in the Timp-3–deficient mammary gland

    OpenAIRE

    Fata, Jimmie E.; Leco, Kevin J.; Voura, Evelyn B.; Hoi-Ying E Yu; Waterhouse, Paul; Murphy, Gillian; Moorehead, Roger A.; Khokha, Rama

    2001-01-01

    The proapoptotic proteinase inhibitor TIMP-3 is the only molecule of this family thought to influence cell death. We examined epithelial apoptosis in TIMP-3–deficient mice during mammary gland involution. Lactation was not affected by the absence of TIMP-3, but glandular function, as measured by gland-to-body weight ratio and production of β-casein, was suppressed earlier during post-lactational involution than in controls. Histological examination revealed accelerated lumen collapse, alveola...

  4. Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

    Science.gov (United States)

    Stewart, Michael K. G.; Plante, Isabelle; Penuela, Silvia; Laird, Dale W.

    2016-01-01

    Pannexin1 (Panx1) subunits oligomerize to form large-pore channels between the intracellular and extracellular milieu that have been shown to regulate proliferation, differentiation and cell death mechanisms. These key cellular responses are ultimately necessary for normal tissue development and function but the role of Panx1 in development, differentiation and function in many tissues remains unexplored, including that of the breast. Panx1 was identified to be expressed in the mammary gland through western blot and immunofluorescent analysis and is dynamically upregulated during pregnancy and lactation. In order to evaluate the role of Panx1 in the context of mammary gland development and function, Panx1-/- mice were evaluated in comparison to wild-type mice in the mammary glands of virgin, lactating and involuting mice. Our results revealed that Panx1 ablation did not affect virgin or involuting mammary glands following histological and whole mount analysis. Panx1 was necessary for timely alveolar development during early lactation based on a decreased number of alveolar lumen following histological analysis and reduced proliferation following Ki67 immunofluorescent labelling. Importantly, the loss of Panx1 in lactating mammary glands did not overtly affect epithelial or secretory differentiation of the mammary gland suggesting that Panx1 is not critical in normal mammary gland function. In addition, PANX1 mRNA expression was correlated with negative clinical outcomes in patients with breast cancer using in silico arrays. Together, our results suggest that Panx1 is necessary for timely alveolar development following the transition from pregnancy to lactation, which may have implications extending to patients with breast cancer. PMID:27099931

  5. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M.; Mroue, Rana; Spencer, Virginia A.; Brownfield, Doug; Radisky, Derek C.; Bustamante, Carlos; Bissell, Mina J.

    2008-10-20

    In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

  6. Antiviral effects of bovine interferons on bovine respiratory tract viruses.

    OpenAIRE

    Fulton, R W; Downing, M M; Cummins, J M

    1984-01-01

    The antiviral effects of bovine interferons on the replication of bovine respiratory tract viruses were studied. Bovine turbinate monolayer cultures were treated with bovine interferons and challenged with several bovine herpesvirus 1 strains, bovine viral diarrhea virus, parainfluenza type 3 virus, goat respiratory syncytial virus, bovine respiratory syncytial virus, bovine adenovirus type 7, or vesicular stomatitis virus. Treatment with bovine interferons reduced viral yield for each of the...

  7. Effect of bisphenol A on morphology, apoptosis and proliferation in the resting mammary gland of the adult albino rat.

    Science.gov (United States)

    Ibrahim, Marwa A A; Elbakry, Reda H; Bayomy, Naglaa A

    2016-02-01

    Bisphenol A (BPA) is a synthetic oestrogen that is extensively used in a wide range of daily used plastic products. This makes it one of the environmental chemicals that may have impact on human health. Due to its oestrogenic effect, BPA might affect the mammary gland. This study aimed to investigate the influence of BPA on the histological structure of the mammary gland of the adult female albino rat and its effect on epithelial cell proliferation and apoptosis status, in addition to its possible modulating effect on estrogen receptor expression. Thirty female adult albino rats were divided into control and experimental groups. The rats in the experimental group were gavaged with 5 mg/kg BPA daily for 8 weeks. The mammary glands were dissected and processed for histological and immunohistochemical stains for Ki-67, activated caspase-3 and estrogen receptor alpha (ER-α). BPA induced an increase in the number and size of the acini and ducts in the mammary gland of treated rats with hyperplasia of their lining epithelial cells. The collagen fibre content was significantly increased in the connective tissue stroma separating the ducts. Immunohistochemical results showed a significant increase in Ki-67 and caspase-3, but a non-significant increase in ER-α expression. Bisphenol A induced structural changes and affected the proliferation rate of mammary glands, so it might be one of the predisposing factors for breast cancer. PMID:26877094

  8. Quantification of mammary organoid toxicant response and mammary tissue motility using OCT fluctuation spectroscopy (Conference Presentation)

    Science.gov (United States)

    Yu, Xiao; Blackmon, Richard L.; Carabas-Hernendez, Patricia; Fuller, Ashley; Troester, Melissa A.; Oldenburg, Amy L.

    2016-03-01

    Mammary epithelial cell (MEC) organoids in 3D culture recapitulate features of breast ducts in vivo. OCT has the ability to monitor the evolution of MEC organoids non-invasively and longitudinally. The anti-cancer drug Doxorubicin (Dox) is able to inhibit proliferation of cancer cells and has been widely used for chemotherapy of breast cancers; while environmental toxins implicated in breast cancer such as estrogen regulates mammary tumor growth and stimulates the proliferation and metastatic potential of breast cancers. Here we propose a quantitative method for measuring motility of breast cells in 3D cultures based upon OCT speckle fluctuation spectroscopy. The metrics of the inverse power-law exponent (α) and fractional modulation amplitude (M) were extracted from speckle fluctuation spectra. These were used to quantify the responses of MEC organoids to Dox, and estrogen. We investigated MEC organoids comprised of two different MEC lines: MCF10DCIS.com exposed to Dox, and MCF7 exposed to estrogen. We found an increase (p<0.001) in α of MEC along time (t=0, 1 hour, 24 hours, 48 hours and 6 days) at each dose of Dox (0, 1 μM and 10 μM), indicating lower fluctuation intensity at higher frequencies. We also observed a decrease (p<0.001) in M for increasing time. However, both α and M of MCF7 treated with estrogen (0, 1 nM and 10 nM) exhibited the opposite trend along time. This novel technology provides rapid and non-invasive measurements of the effects of toxicants on MEC motility for understanding breast cancer development and assessing anti-cancer drugs.

  9. 人凝血因子Ⅸ乳腺特异表达载体的构建及细胞转染%Construction of Human CoaguLation Factor Ⅸ Mammary Expression Vector and Transfection

    Institute of Scientific and Technical Information of China (English)

    韩雪洁; 萨日娜; 梁浩; 李雪玲; 李荣凤

    2014-01-01

    [Objective]Human coagulation factor Ⅸ (hFIX) plays a key role in blood coagulation and is important for clinical treatment of hemophilia. The objective of this study is to construct human coagulation factor Ⅸ (hFIX) mammary expression vector and test the expression of hFIX in porcine mammary epithelial, and obtain hFIX-transgenic porcine mammary epithelial and female porcine fetal fibroblast cells to prepare to produce hFIX mammary specific expression transgenic pigs .[Method]Total RNA was extracted from human fetal liver tissues and human coagulation factor Ⅸ (hFIX) cDNA was amplified by RT-PCR followed the instructions of RNAiso Reagent and Prime Script RT-PCR Kit from TAKARA. PCR was performed to amplify bovine growth hormone (BGH) polyA fragment. Both hFIX cDNA and BGH polyA fragments were cloned to pbCSN2-RC plasmid, located after the bovine beta-Casein (CSN2) promoter, to achieve mammary specific expression vector pbCSN2-hFIX-pA-RC with neomycin-resistance and red fluorescence genes. Porcine mammary epithelial cells were obtained by culturing 1mm3 mammary tissue cubes from the breast tissue of lactation pigs that slaughtered in the local slaughterhouse and purifying with time-controlled trypsinization. The chromosome analysis was performed on the derived cells, and the cells with normal number of chromosomes were transfected with pbCSN2-hFIX-pA-RC by lipofection technology. The transfected porcine mammary epithelial cells were screened by neomycin-resistance and red fluorescent expression. The expression of hFIX was further confirmed by real-time PCR. To only transfect the female fibroblasts, the SRY PCR was performed on the porcine fibroblasts cultured in the lab to determine the sex of the cells. Finally, the confirmed vector was transferred to porcine fetal fibroblast cells by lipofection technology. [Result] The results of PCR and restricted endonucleases digestion analysis showed that the hFIX cDNA and BGH PolyA were cloned into pbCSN2-RC, and the

  10. Paternal selenium deficiency but not supplementation during preconception alters mammary gland development and 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in female rat offspring.

    Science.gov (United States)

    Guido, Luiza N; Fontelles, Camile C; Rosim, Mariana P; Pires, Vanessa C; Cozzolino, Silvia M F; Castro, Inar A; Bolaños-Jiménez, Francisco; Barbisan, Luis F; Ong, Thomas P

    2016-10-15

    Breast cancer is a global public health problem and accumulating evidence indicates early-life exposures as relevant factors in the disease risk determination. Recent studies have shown that paternal nutrition can influence offspring health including breast cancer risk. Selenium is a micronutrient with essential role in central aspects of embryogenesis, male fertility and cancer and that has been extensively studied as a chemopreventive agent in several breast cancer experimental models. Thus, we designed an animal study to evaluate whether paternal selenium deficiency or supplementation during preconception could affect the female offspring mammary gland development and breast cancer susceptibility. Male Sprague-Dawley rats were fed AIN93-G diet containing 0.15 ppm (control diet), 0.05 ppm (deficient diet) or 1 ppm (supplemented diet) of selenium for 9 weeks and mated with control female rats. Mammary carcinogenesis was induced with 7,12-dimethylbenz[a]anthracene (DMBA) in their female offspring. Paternal selenium deficiency increased the number of terminal end buds, epithelial elongation and cell proliferation in the mammary gland of the female rat offspring and these effects were associated with higher susceptibility to DMBA-induced mammary tumors (increased incidence and higher grade tumors). On the other hand, paternal selenium supplementation did not influence any of these parameters. These results highlight the importance of father's nutrition including selenium status as a relevant factor affecting daughter's breast cancer risk and paternal preconception as a potential developmental stage to start disease preventive strategies. PMID:27270969

  11. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells

    Directory of Open Access Journals (Sweden)

    Arantzazu eZubeldia-Plazaola

    2015-05-01

    Full Text Available Two lineages, epithelial and myoepithelial cells, are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells and CD10/K14 (myoepithelial cells antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast

  12. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

    Science.gov (United States)

    Zubeldia-Plazaola, Arantzazu; Ametller, Elisabet; Mancino, Mario; Prats de Puig, Miquel; López-Plana, Anna; Guzman, Flavia; Vinyals, Laia; Pastor-Arroyo, Eva M; Almendro, Vanessa; Fuster, Gemma; Gascón, Pedro

    2015-01-01

    Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer. PMID

  13. Tumour-stromal interactions: Reciprocal regulation of extracellular matrix proteins and ovarian steroid activity in the mammary gland

    International Nuclear Information System (INIS)

    Despite the critical importance of ovarian steroids in the treatment of breast cancer, little is known about the acquisition or loss of estrogen and progesterone responsiveness in either the normal or neoplastic mammary gland. This review focuses on the interactions among mammary stroma-derived extracellular matrix (ECM) proteins, integrins and ovarian hormone-dependent proliferation in normal and neoplastic mammary cells both in vivo and in vitro. In vitro studies show that fibronectin is required for progesterone-induced proliferation of normal mammary epithelial cells and that specific ECM proteins also regulate interactions between growth factors and ovarian hormones. Studies with human breast cancer cell lines have shown that laminin inhibits estrogen-induced proliferation and estrogen-response-element-mediated transcription in vitro and also inhibits estrogen-induced proliferation in vivo. Reciprocally, ovarian steroids regulate the expression of ECM proteins and their cellular receptors, integrins, during mammary gland development in vivo. The fibronectin-specific integrin, α5β1 is regulated by ovarian steroids and its expression is positively correlated with developmental stages of peak proliferation. These studies suggest that the coordinated regulation of ovarian hormone responsiveness and ECM/integrin expression may be critical to normal mammary gland development and breast cancer growth and progression

  14. Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis

    Directory of Open Access Journals (Sweden)

    Cassali Geovanni D

    2010-02-01

    Full Text Available Abstract Background It has been suggested that columnar cell lesions indicate an alteration of the human mammary gland involved in the development of breast cancer. They have not previously been described in canine mammary gland. The aim of this paper is describe the morphologic spectrum of columnar cell lesions in canine mammary gland specimens and their association with other breast lesions. Methods A total of 126 lesions were subjected to a comprehensive morphological review based upon the human breast classification system for columnar cell lesions. The presence of preinvasive (epithelial hyperplasia and in situ carcinoma and invasive lesions was determined and immunophenotypic analysis (estrogen receptor (ER, progesterone receptor (PgR, high molecular weight cytokeratin (34βE-12, E-cadherin, Ki-67, HER-2 and P53 was perfomed. Results Columnar cell lesions were identified in 67 (53.1% of the 126 canine mammary glands with intraepithelial alterations. They were observed in the terminal duct lobular units and characterized at dilated acini may be lined by several layers of columnar epithelial cells with elongated nuclei. Of the columnar cell lesions identified, 41 (61.2% were without and 26 (38.8% with atypia. Association with ductal hyperplasia was observed in 45/67 (67.1%. Sixty (89.5% of the columnar cell lesions coexisted with neoplastic lesions (20 in situ carcinomas, 19 invasive carcinomas and 21 benign tumors. The columnar cells were ER, PgR and E-cadherin positive but negative for cytokeratin 34βE-12, HER-2 and P53. The proliferation rate as measured by Ki-67 appeared higher in the lesions analyzed than in normal TDLUs. Conclusions Columnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions.

  15. Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis

    International Nuclear Information System (INIS)

    It has been suggested that columnar cell lesions indicate an alteration of the human mammary gland involved in the development of breast cancer. They have not previously been described in canine mammary gland. The aim of this paper is describe the morphologic spectrum of columnar cell lesions in canine mammary gland specimens and their association with other breast lesions. A total of 126 lesions were subjected to a comprehensive morphological review based upon the human breast classification system for columnar cell lesions. The presence of preinvasive (epithelial hyperplasia and in situ carcinoma) and invasive lesions was determined and immunophenotypic analysis (estrogen receptor (ER), progesterone receptor (PgR), high molecular weight cytokeratin (34βE-12), E-cadherin, Ki-67, HER-2 and P53) was perfomed. Columnar cell lesions were identified in 67 (53.1%) of the 126 canine mammary glands with intraepithelial alterations. They were observed in the terminal duct lobular units and characterized at dilated acini may be lined by several layers of columnar epithelial cells with elongated nuclei. Of the columnar cell lesions identified, 41 (61.2%) were without and 26 (38.8%) with atypia. Association with ductal hyperplasia was observed in 45/67 (67.1%). Sixty (89.5%) of the columnar cell lesions coexisted with neoplastic lesions (20 in situ carcinomas, 19 invasive carcinomas and 21 benign tumors). The columnar cells were ER, PgR and E-cadherin positive but negative for cytokeratin 34βE-12, HER-2 and P53. The proliferation rate as measured by Ki-67 appeared higher in the lesions analyzed than in normal TDLUs. Columnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions

  16. Canine mammary tumours, an overview.

    Science.gov (United States)

    Sleeckx, N; de Rooster, H; Veldhuis Kroeze, E J B; Van Ginneken, C; Van Brantegem, L

    2011-12-01

    Canine mammary tumours (CMTs) are the most common neoplasms in intact female dogs. Although the prevalence of these tumours decreases in regions where preventive ovari(ohyster)ectomy is performed, it remains an important disease entity in veterinary medicine. Moreover, treatment options are limited in comparison with human breast cancer. Nevertheless, recent human treatment protocols might have potential in bitches suffering from CMTs. PMID:21645126

  17. Ectopic mammary tissue in vulva

    Directory of Open Access Journals (Sweden)

    Đorđević Momčilo

    2008-01-01

    Full Text Available Background. Ectopic mammary gland tissue is a residual tissue that persists during the embryologic development along ectodermal primitive milk streaks. Incomplete involution anywhere along the primitive milk streak can result in accessory or ectopic mammary tissue. Case report. A woman, 27-year old, admitted to Obstetrics and Gynecology Clinic Kragujevac for surgery, of goose-egg size, vulva tumor, of elastic consistency. Menarche started in 12 years of age, with the regular menstrual cycle, without previous gynecological diseases. The woman had one pregnancy terminated by cesarean section because of the multiple (twin pregnancy. Excision of the tumor was completely done in the total endotracheal anesthesia. Pathohistologic (PH findings was: Dysplasia fibrosa cystica simplex mammae, with focuses of sclerosing adenosis. Expression of estrogen (ER and progesterone receptors (PR were positive. Conclusion. Ectopic mammary tissue in vulva in adult period is very rarely seen, and can be changed pathologically as well as normally positioned breast tissue into benign cystic changes, benign tumors, adenomas and fibroadenomas and tumors. Cells with low ER/PR receptor level grow independently of estrogene stimulation and they could be resistant to hormonal therapy effects.

  18. Mammary neoplasms of the bitch.

    Science.gov (United States)

    Cotchin, E

    1958-01-01

    In this paper, the interrelationships of the neoplasms of the canine mammary gland are investigated. These neoplasms are a group of tumors of a great variety of histological structure and sometimes of uncertain histogenesis. Particular attention is given to the histogenesis of the mucoid, cartilaginous, and bony elements. From 1950-56, a macroscopic and histological examination of mammary neoplasms from 424 bitches (2-17 years of age) was made. The tumors from 381 bitches were removed surgically while the others came from 43 bitches who were examined postmortem. Of the 160 tumors whose location was recorded, 105 occurred in the 2 hinder glands, 19 in the middle glands, and 46 in one or another of the 2 anterior glands. 186 of the 424 bitches bore malignant mammary tumors (87 carcinomas, 73 sarcomas, 27 complex malignant tumors) and 249 had benign tumors (19 simple and 230 complex). 40 of the benign complex tumors contained bone, an additional 63 contained cartilage but no bone, and 67 showed mucoid tissue but no cartilage or bone. It is suggested that there is a predominant proliferation of myoepithelial cells which tend to become embedded in a mucoid or chondroid matrix. The bone in the tumors appears to be formed by endochondral ossification of preformed cartilage, or by intramembranous ossification in the connective tissue of the tumor. Metastases were present in 41 of the 424 bitches. PMID:12311486

  19. A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland

    International Nuclear Information System (INIS)

    This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine β-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland

  20. Inhibition of proliferation by PERK regulates mammary acinar morphogenesis and tumor formation.

    Directory of Open Access Journals (Sweden)

    Sharon J Sequeira

    Full Text Available Endoplasmic reticulum (ER stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2alpha. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2alpha and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-DeltaC or PERK-K618A, does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKDeltaC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.

  1. Radiogenic neoplasia in thyroid and mammary clonogens

    International Nuclear Information System (INIS)

    We have developed rat thyroid and mammary clonogen transplantation systems for the study of radiogenic cancer induction at the target cell level in vivo. The epithelial cell populations of both glands contain small subpopulations of cells which are capable of giving rise to monoclonal glandular structures when transplanted and stimulated with appropriate hormones. Previous results indicated that these clonogens are the precursor cells of radiogenic cancer, and that initiation, is common event at the clonegenic cell level. Detailed information on the physiologic control of clonogen proliferation, differentiation, and total numbers is thus essential to an understanding of the carcinogenic process. We report here studies on investigations on the relationships between grafted thyroid cell number and the rapidity and degree of reestablishment of the thyroid-hypothalamus-pituitary feedback axis in thyroidectomized rats maintained on a normal diet or an iodine deficient diet; studies of the persistence of, and the differentiation potential and functional characteristics of, the TSH-(thyrotropin-) responsive sub- population of clonogens during goitrogenesis, the plateau-phase of goiter growth, and goiter involution; studies of changes in the size of the clonogen sub-population during goitrogenesis, goiter involution and the response to goitrogen rechallenge; and a large carcinogenesis experiment on the nature of the grafted thyroid cell number-dependent suppression of promotion/progression to neoplasia in grafts of radiation-initiated thyroid cells. Data from these studies will be used in the design of future carcinogenesis experiments on neoplastic initiation by high and low LET radiations and on cell interactions during the neoplastic process

  2. Innate immune response to a bovine mastitis pathogen profiled in milk and blood monocytes using a systems biology approach

    Science.gov (United States)

    Bovine mastitis is an inflammatory condition of the mammary gland which leads to reduced milk yield and increased milk somatic cell counts (SCC) resulting in an estimated annual cost to the dairy industry worldwide of ~ 2 billion euros. Mastitis has a complex etiology, with pathogenic, host and envi...

  3. Prepubertal unilateral gynecomastia and the presence of 47,XXY mosaicism in breast epithelial cells

    DEFF Research Database (Denmark)

    Andersen, Peter Stemann; Petersen, Bodil Laub; Juul, Anders;

    2013-01-01

    sclerotherapy with Picibanil (OK-432) was attempted without any detectable effect on size. The mass was later excised. The pathological examination revealed mammary gland tissue suggestive of idiopathic gynecomastia. FISH revealed 47, XXY mosaicism in the abnormal breast epithelial cells, but not in peripheral...

  4. A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development.

    Science.gov (United States)

    Kitayama, Midori; Mizutani, Kiyohito; Maruoka, Masahiro; Mandai, Kenji; Sakakibara, Shotaro; Ueda, Yuki; Komori, Takahide; Shimono, Yohei; Takai, Yoshimi

    2016-03-11

    Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development. PMID:26757815

  5. Mammary Hypertrophy in an Ovariohysterectomized Cat

    OpenAIRE

    Pukay, B.P.; Stevenson, D.A.

    1983-01-01

    A four year old ovariohysterectomized domestic short-haired cat under treatment for behavioral urine spraying and idiopathic alopecia developed mammary gland hypertrophy following treatment with megestrol acetate. Withdrawal of the progestin and treatment with androgen failed to cause regression of the hypertrophy. The affected mammary gland was surgically excised and recovery was uneventful.

  6. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY... live bovines and products derived from bovines with regard to bovine spongiform encephalopathy. This.... SUPPLEMENTARY INFORMATION: On March 16, 2012, we published in the Federal Register (77 FR 15848-15913, Docket...

  7. Emergence of nuclear heparanase induces differentiation of human mammary cancer cells

    International Nuclear Information System (INIS)

    The study of epithelial differentiation touches upon many modern aspects of biology. The epithelium is in constant dialogue with the underlying mesenchyme to control stem cell activity, proliferation in transit-amplifying compartments, lineage commitment, terminal differentiation and, ultimately, cell death. There are spatially distinct compartments dedicated to each of these events. Recently we reported that heparanase is expressed in nucleus as well as in the cytoplasm and that nuclear heparanase seems to be related to cell differentiation. In this study, we investigated the role of nuclear heparanase in differentiation by transducing human mammary epithelial cancer cells with heparanase which was delivered specifically into nucleus. We observed that expression of nuclear heparanase allowed the cells to differentiate with the appearance of lipid droplets. This finding supports the idea that heparanase plays a novel role in epithelial cell differentiation apart from its known enzymatic function

  8. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  9. Effect of protein quality on 14C glucose utilization in isolated rat mammary acini

    International Nuclear Information System (INIS)

    Poor protein quality has a deleterious effect on lactation in rats. Dams consuming a 13% wheat gluten (WG) diet are unable to maintain litters. Glucose utilization in isolated mammary acini taken from dams at either day 20 of gestation (G20) or day 4 of lactation (L4) was examined in dams consuming 13% WG vs 13% casein-methionine (CM) diets from day of breeding. Dams consuming WG had significantly smaller inguinal-abdominal mammary glands than CM dams at both G20 and L4, and mammary glands of CM but not WG dams were larger at L4 than G20. Both average pup weight and pup daily gain were smaller in WG litters. Basal levels of 14C glucose oxidation (GO) and 14C glucose incorporation into lipid (GL) and lactose were examined. A large significant increase in GO and GL occurred in CM dams from G20 to L4 but not in WG dams. Both GO and GL were higher in CM dams on L4 but not at G20. The ratio of GO:GO+GL changed at parturition in CM but not WG dams. The normal changes in glucose utilization by mammary epithelial cells which occur at parturition were impaired by the WG diet

  10. CCAAT/enhancer binding proteins in normal mammary development and breast cancer

    International Nuclear Information System (INIS)

    CCAAT/enhancer binding proteins (C/EBPs) are a family of leucine zipper, transcription factors that bind to DNA as homodimers and heterodimers. They regulate cellular proliferation, differentiation and apoptosis in the mammary gland. Multiple protein isoforms, including truncated, dominant negatives, are generated by translation of the C/EBPβ transcript or via proteolytic cleavage of the full-length C/EBPβ protein. Gene deletion of individual C/EBP family members has demonstrated an essential role for C/EBPβ in normal mammary development, while transgenic and overexpression studies provide evidence that the dominant-negative C/EBPβ-liver-enriched inhibitory protein isoform induces proliferation in mammary epithelial cells. Mounting evidence suggests that alterations in the ratio of the C/EBPβ-liver-enriched inhibitory protein isoform and the C/EBPβ-liver-enriched activating protein isoform may play a role in the development of breast cancer. This review will consequently focus on C/EBP actions in normal mammary development and on the emerging data that supports a role in breast cancer

  11. A Progesterone-CXCR4 Axis Controls Mammary Progenitor Cell Fate in the Adult Gland

    Directory of Open Access Journals (Sweden)

    Yu-Jia Shiah

    2015-03-01

    Full Text Available Progesterone drives mammary stem and progenitor cell dynamics through paracrine mechanisms that are currently not well understood. Here, we demonstrate that CXCR4, the receptor for stromal-derived factor 1 (SDF-1; CXC12, is a crucial instructor of hormone-induced mammary stem and progenitor cell function. Progesterone elicits specific changes in the transcriptome of basal and luminal mammary epithelial populations, where CXCL12 and CXCR4 represent a putative ligand-receptor pair. In situ, CXCL12 localizes to progesterone-receptor-positive luminal cells, whereas CXCR4 is induced in both basal and luminal compartments in a progesterone-dependent manner. Pharmacological inhibition of CXCR4 signaling abrogates progesterone-directed expansion of basal (CD24+CD49fhi and luminal (CD24+CD49flo subsets. This is accompanied by a marked reduction in CD49b+SCA-1− luminal progenitors, their functional capacity, and lobuloalveologenesis. These findings uncover CXCL12 and CXCR4 as novel paracrine effectors of hormone signaling in the adult mammary gland, and present a new avenue for potentially targeting progenitor cell growth and malignant transformation in breast cancer.

  12. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, Yohei; Radisky, Derek; Boudreau, Rosanne; Simian, Marina; Stevens, Mary E.; Oka, Yumiko; Takebe, Kyoko; Niwa, Shinichiro; Bissell, Mina J.

    2002-03-22

    We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression.

  13. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta

    International Nuclear Information System (INIS)

    We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression

  14. Differential expression of serotonin, tryptophan hydroxylase and monoamine oxidase A in the mammary gland of the Myotis velifer bat.

    Directory of Open Access Journals (Sweden)

    Cristián Vela Hinojosa

    Full Text Available The mammary gland has long drawn the attention of the scientific community due to the limited knowledge of some fundamental aspects involved in the control of its function. Myotis velifer, a microchiropteran species, provides an interesting model to study some of the regulatory factors involved in the control of the mammary gland cycle. Having an asynchronous, monoestrous reproductive pattern, female M. velifer bats undergo drastic morphological changes of the breast during the reproductive cycle. Current research on non-chiropteran mammals indicates that serotonin (5-HT plays a major role in the intraluminal volume homeostasis of the mammary gland during lactation; however, an analysis of both the expression and localization of the main components of the serotonergic system in the bat mammary gland is lacking. Thus, the objectives of the present study were: to describe the gross and histological anatomy of the mammary gland of M. velifer to establish the lactation period for this species; to analyze the distribution and expression of the main serotonergic components in the mammary tissues of these bats under the physiological conditions of lactation, involution and the resting phase; and to provide information on the involvement of 5-HT in the regulation of the physiological function of this organ. To assess the expression and localization of serotonergic components, multiple immunofluorescence, Western blot and HPLC methods were used. 5-HT and the enzyme that catalyzes its synthesis (TPH were located in both myoepithelial and luminal epithelial cells, while the enzyme responsible for the catabolism of this neurohormone (MAO A was found in luminal epithelial cells as well as in secreted products. We also found an increased expression of serotonergic components during lactation, indicating that elements of the serotonergic system may play an important role in lactation in this species of bat in a way similar to that of other mammal species.

  15. Differential Expression of Serotonin, Tryptophan Hydroxylase and Monoamine Oxidase A in the Mammary Gland of the Myotis velifer Bat

    Science.gov (United States)

    Vela Hinojosa, Cristián; León Galván, Miguel Angel; Tapia Rodríguez, Miguel; López Ortega, Gerardo; Cerbón Cervantes, Marco Antonio; Rodríguez, Carmen Adriana Mendoza; Cortés, Patricia Padilla; Méndez, Luis Antonio Martínez; Trejo, Francisco Javier Jiménez

    2013-01-01

    The mammary gland has long drawn the attention of the scientific community due to the limited knowledge of some fundamental aspects involved in the control of its function. Myotis velifer, a microchiropteran species, provides an interesting model to study some of the regulatory factors involved in the control of the mammary gland cycle. Having an asynchronous, monoestrous reproductive pattern, female M. velifer bats undergo drastic morphological changes of the breast during the reproductive cycle. Current research on non-chiropteran mammals indicates that serotonin (5-HT) plays a major role in the intraluminal volume homeostasis of the mammary gland during lactation; however, an analysis of both the expression and localization of the main components of the serotonergic system in the bat mammary gland is lacking. Thus, the objectives of the present study were: to describe the gross and histological anatomy of the mammary gland of M. velifer to establish the lactation period for this species; to analyze the distribution and expression of the main serotonergic components in the mammary tissues of these bats under the physiological conditions of lactation, involution and the resting phase; and to provide information on the involvement of 5-HT in the regulation of the physiological function of this organ. To assess the expression and localization of serotonergic components, multiple immunofluorescence, Western blot and HPLC methods were used. 5-HT and the enzyme that catalyzes its synthesis (TPH) were located in both myoepithelial and luminal epithelial cells, while the enzyme responsible for the catabolism of this neurohormone (MAO A) was found in luminal epithelial cells as well as in secreted products. We also found an increased expression of serotonergic components during lactation, indicating that elements of the serotonergic system may play an important role in lactation in this species of bat in a way similar to that of other mammal species. PMID:24086437

  16. Thymic and mammary lymphosarcoma in a three-year-old heifer.

    Science.gov (United States)

    Matthews, H K; Hunt, E; Duncan, D E

    1992-03-01

    Ventral edema, dyspnea, fever, tachycardia, bloat and muffled heart sounds were identified in a 3-year-old heifer. Attempts to relieve the bloat by passing an orogastric tube were unsuccessful. The heifer was bovine leukemia virus-negative by the agar gel immunodiffusion test, and had normocytic, normochromic anemia, mature neutrophilia, and hyperproteinemia. Pleural effusion was identified by thoracic ultrasonography. Cytologic examination of pleural fluid revealed an increased number of atypical lymphocytes. The heifer died, and at necropsy, thymic and metastatic mammary lymphosarcoma was confirmed. PMID:1568914

  17. TNFalpha-mediated plasminogen activation on neutrophils is involved in the high plasmin activity in mammary secretion of drying-off cows.

    Science.gov (United States)

    Chou, Wen K; Yu, Ting C; Chen, Shuen E; Peh, Ho C; Liu, Wen B; Chen, Ming T; Nagahata, Hajime; Chang, Chai J

    2009-11-01

    Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-alpha in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (Pm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-alpha antibody, and was further up-regulated by exogenous bovine TNF-alpha. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-alpha decreased immediately following milk stasis. Iimmunoblotting revealed that although both 26-kDa pro-TNF-alpha and 17-kDa TNF-alpha were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-alpha was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-alpha-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-alpha on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-alpha level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary

  18. Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats

    Directory of Open Access Journals (Sweden)

    Whitsett Timothy

    2006-05-01

    Full Text Available Abstract Despite the advent of new and aggressive therapeutics, breast cancer remains a leading killer among women; hence there is a need for the prevention of this disease. Several naturally occurring polyphenols have received much attention for their health benefits, including anti-carcinogenic properties. Two of these are resveratrol, a component of red grapes, and epigallocatechin-3-gallate (EGCG, the major catechin found in green tea. In this study, we tested the hypothesis that these two polyphenols protect against chemically-induced mammary cancer by modulating mammary gland architecture, cell proliferation, and apoptosis. Female Sprague-Dawley CD rats were exposed to either resveratrol (1 g/kg AIN-76A diet, EGCG (0.065% in the drinking water, or control diet (AIN-76A for the entirety of their life starting at birth. At 50 days postpartum, rats were treated with 60 mg dimethylbenz[a]anthracene (DMBA/kg body weight to induce mammary cancer. Resveratrol, but not EGCG, suppressed mammary carcinogenesis (fewer tumors per rat and longer tumor latency. Analysis of mammary whole mounts from 50-day-old rats revealed that resveratrol, but not EGCG, treatment resulted in more differentiated lobular structures. Bromodeoxyuridine (BrdU incorporation studies showed that resveratrol treatment caused a significant reduction in proliferative cells in mammary terminal ductal structures at 50 days postpartum, making them less susceptible to carcinogen insult. The epithelial cells of terminal end buds in the mammary glands of resveratrol-treated rats also showed an increase in apoptotic cells compared to the control or EGCG-treated rats as measured by a DNA fragmentation assay. At the given doses, resveratrol treatment resulted in a serum resveratrol concentration of 2.00 μM, while treatment with EGCG resulted in a serum EGCG concentration of 31.06 nM. 17β-Estradiol, progesterone, and prolactin concentrations in the serum were not significantly affected

  19. Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium

    International Nuclear Information System (INIS)

    Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purifed and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a λgt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven crossreacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI

  20. The antiprogestins mifepristone and onapristone reduce cell proliferation in the canine mammary carcinoma cell line CMT-U27.

    Science.gov (United States)

    Guil-Luna, Silvia; Hellmén, Eva; Sánchez-Céspedes, Raquel; Millán, Yolanda; Martín de las Mulas, Juana

    2014-07-01

    Canine mammary tumours (CMTs) represent nearly half of all tumours in female dogs and some 50% have malignant behaviour. Simple epithelial carcinomas have shorter disease free periods after surgery and a higher reduction of the proliferation index reduction after antiprogestin aglepristone treatment in vivo related to the expression of progesterone receptors (PR). These findings make simple carcinomas good candidates for endocrine therapy. To further explore this possibility, the effects of the antiprogestins mifepristone (RU486) and onapristone (ZK299) on cell viability and PR expression of the canine mammary carcinoma cell line isolated from a simple epithelial carcinoma CMT-U27 were studied. Twenty five percent of CMT-U27 control cells expressed PR. RU486 (ptest) at 24h but only the latter treatment reduced significantly PR expression in viable tumour cells at 24h of incubation. The results suggest that both RU486 and ZK299 induce a decrease in the number of viable CMT-U27 tumour cells with different effects on PR expression. The canine mammary carcinoma cell line CMT-U27 is sensitive to the effects of antiprogestins and may serve to further explore the role of these drugs in canine mammary carcinomas. PMID:24500783

  1. In vitro evaluation of a novel bacteriophage cocktail as a preventative for bovine coliform mastitis.

    Science.gov (United States)

    Porter, J; Anderson, J; Carter, L; Donjacour, E; Paros, M

    2016-03-01

    The objective of this study was to investigate the potential use of bacteriophage in preventing Escherichia coli mastitis on dairies. A cocktail consisting of 4 distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a relatively broad spectrum of action against relevant strains from 2 distinct geographies. The ability to suppress bacterial growth of these isolates in a liquid growth medium was not affected by the ratio of bacteriophage particles to bacterial cells (multiplicity of infection, MOI). For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10had the same effect as 10µg/mL of ceftiofur on the growth rate of E. coli over a 12-h period using optical density measurements. A 3.3- to 5.6-log reduction of growth was achieved when E. coli was co-incubated with our phage cocktail in raw milk over a 12-h period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pretreatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli compared with controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6×10(3) cfu/mL of a clinical mastitis E. coli strain. In vitro results show bactericidal activity by our phage in raw milk and mammary tissue culture systems. Before a bacteriophage-based dry-cow treatment becomes a potential option for dairies, in vivo studies must be able to demonstrate that a specific dose of bacteriophage can protect cows from

  2. Mammary and extramammary Paget's disease*

    Science.gov (United States)

    Lopes, Lauro Lourival; Lopes, Ione Maria Ribeiro Soares; Lopes, Lauro Rodolpho Soares; Enokihara, Milvia M. S. S.; Michalany, Alexandre Osores; Matsunaga, Nobuo

    2015-01-01

    Paget's disease, described by Sir James Paget in 1874, is classified as mammary and extramammary. The mammary type is rare and often associated with intraductal cancer (93-100% of cases). It is more prevalent in postmenopausal women and it appears as an eczematoid, erythematous, moist or crusted lesion, with or without fine scaling, infiltration and inversion of the nipple. It must be distinguished from erosive adenomatosis of the nipple, cutaneous extension of breast carcinoma, psoriasis, atopic dermatitis, contact dermatitis, chronic eczema, lactiferous ducts ectasia, Bowen's disease, basal cell carcinoma, melanoma and intraductal papilloma. Diagnosis is histological and prognosis and treatment depend on the type of underlying breast cancer. Extramammary Paget's disease is considered an adenocarcinoma originating from the skin or skin appendages in areas with apocrine glands. The primary location is the vulvar area, followed by the perianal region, scrotum, penis and axillae. It starts as an erythematous plaque of indolent growth, with well-defined edges, fine scaling, excoriations, exulcerations and lichenification. In most cases it is not associated with cancer, although there are publications linking it to tumors of the vulva, vagina, cervix and corpus uteri, bladder, ovary, gallbladder, liver, breast, colon and rectum. Differential diagnoses are candidiasis, psoriasis and chronic lichen simplex. Histopathology confirms the diagnosis. Before treatment begins, associated malignancies should be investigated. Surgical excision and micrographic surgery are the best treatment options, although recurrences are frequent. PMID:25830993

  3. Breast metastases primitive extra mammary

    International Nuclear Information System (INIS)

    Less than 3% of all breast cancers originate from a primitive extra mammary. In 40% of cases it is the first manifestation of the primitive properly studied but 80% are associated with widely disseminated disease. It typically presents as a nodule on external quadrant s painful in half the cases. The majority (60%) of metastases derived from breast contralateral breast tumors are believed to via the lymphatic system. of the ; extra mammary the most common tumors are melanoma; hematologic and neuroendocrine. Although some imaging characteristics can guide diagnosis is histological. Cytology has good performance in experienced hands; but up to 25% of cases there may be difficulty in establishing diagnosis. Treatment depends on the type of tumor. Mastectomy should not be practiced or axillary clearance routine as is generally the context of disease disseminated. Radiation therapy may be useful for local control. It has been proposed laser ablation but no experience with it. The overall prognosis is bad. For a man of 45 with a breast metastasis occurs only a clear cell carcinoma of the kidney

  4. Mammary and extramammary Paget's disease.

    Science.gov (United States)

    Lopes Filho, Lauro Lourival; Lopes, Ione Maria Ribeiro Soares; Lopes, Lauro Rodolpho Soares; Enokihara, Milvia M S S; Michalany, Alexandre Osores; Matsunaga, Nobuo

    2015-01-01

    Paget's disease, described by Sir James Paget in 1874, is classified as mammary and extramammary. The mammary type is rare and often associated with intraductal cancer (93-100% of cases). It is more prevalent in postmenopausal women and it appears as an eczematoid, erythematous, moist or crusted lesion, with or without fine scaling, infiltration and inversion of the nipple. It must be distinguished from erosive adenomatosis of the nipple, cutaneous extension of breast carcinoma, psoriasis, atopic dermatitis, contact dermatitis, chronic eczema, lactiferous ducts ectasia, Bowen's disease, basal cell carcinoma, melanoma and intraductal papilloma. Diagnosis is histological and prognosis and treatment depend on the type of underlying breast cancer. Extramammary Paget's disease is considered an adenocarcinoma originating from the skin or skin appendages in areas with apocrine glands. The primary location is the vulvar area, followed by the perianal region, scrotum, penis and axillae. It starts as an erythematous plaque of indolent growth, with well-defined edges, fine scaling, excoriations, exulcerations and lichenification. In most cases it is not associated with cancer, although there are publications linking it to tumors of the vulva, vagina, cervix and corpus uteri, bladder, ovary, gallbladder, liver, breast, colon and rectum. Differential diagnoses are candidiasis, psoriasis and chronic lichen simplex. Histopathology confirms the diagnosis. Before treatment begins, associated malignancies should be investigated. Surgical excision and micrographic surgery are the best treatment options, although recurrences are frequent. PMID:25830993

  5. A Multifunctional 3D Co-Culture System for Studies of Mammary Tissue Morphogenesis and Stem Cell Biology

    OpenAIRE

    Campbell, Jonathan J.; Davidenko, Natalia; Caffarel, Maria M.; Cameron, Ruth E.; Watson, Christine J

    2011-01-01

    Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this sc...

  6. Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling

    OpenAIRE

    Bagci, H; Laurin, M.; Huber, J.; Muller, W J; Côté, J-F

    2014-01-01

    Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) ...

  7. Epstein-Barr Virus, Human Papillomavirus and Mouse Mammary Tumour Virus as Multiple Viruses in Breast Cancer

    OpenAIRE

    Glenn, Wendy K; Heng, Benjamin; Delprado, Warick; Iacopetta, Barry; Whitaker, Noel J.; Lawson, James S.

    2012-01-01

    Background The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. Materials and Methods All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 l...

  8. Expression of Autoactivated Stromelysin-1 in Mammary Glands of Transgenic Mice Leads to a Reactive Stroma During Early Development

    Energy Technology Data Exchange (ETDEWEB)

    Thomasset, N.; Lochter, A.; Sympson, C.J.; Lund, L.R.; Williams, D.R.; Behrendtsen, O.; Werb, Z.; Bissell, M.J.

    1998-04-24

    Extracellular matrix and extracellular matrix-degrading matrix metalloproteinases play a key role in interactions between the epithelium and the mesenchyme during mammary gland development and disease. In patients with breast cancer, the mammary mesenchyme undergoes a stromal reaction, the etiology of which is unknown. We previously showed that targeting of an autoactivating mutant of the matrix metalloproteinase stromelysin-1 to mammary epithelia of transgenic mice resulted in reduced mammary function during pregnancy and development of preneoplastic and neoplastic lesions. Here we examine the cascade of alterations before breast tumor formation in the mammary gland stroma once the expression of the stromelysin-1 transgene commences. Beginning in postpubertal virgin animals, low levels of transgene expression in mammary epithelia led to increased expression of endogenous stromelysin-1 in stromal fibroblasts and up-regulation of other matrix metalloproteinases, without basement membrane disruption. These changes were accompanied by the progressive development of a compensatory reactive stroma, characterized by increased collagen content and vascularization in glands from virgin mice. This remodeling of the gland affected epithelial-mesenchymal communication as indicated by inappropriate expression of tenascin-C starting by day 6 of pregnancy. This, together with increased transgene expression, led to basement membrane disruption starting by day 15 of pregnancy. We propose that the highly reactive stroma provides a prelude to breast epithelial tumors observed in these animals. Epithelial development depends on an exquisite series of inductive and instructive interactions between the differentiating epithelium and the mesenchymal (stromal) compartment. The epithelium, which consists of luminal and myoepithelial cells, is separated from the stroma by a basement membrane (BM), which plays a central role in mammary gland homeostasis and gene expression. In vivo, stromal

  9. Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation.

    Science.gov (United States)

    Lee, Sooyeon; Hennigar, Stephen R; Alam, Samina; Nishida, Keigo; Kelleher, Shannon L

    2015-05-22

    The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women. PMID:25851903

  10. Milk from dams fed an obesogenic diet combined with a high-fat/high-sugar diet induces long-term abnormal mammary gland development in the rabbit.

    Science.gov (United States)

    Hue-Beauvais, C; Koch, E; Chavatte-Palmer, P; Galio, L; Chat, S; Letheule, M; Rousseau-Ralliard, D; Jaffrezic, F; Laloë, D; Aujean, E; Révillion, F; Lhotellier, V; Gertler, A; Devinoy, E; Charlier, M

    2015-04-01

    Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged (P casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function. PMID:26020186

  11. Mouse mammary tumor biology: a short history.

    Science.gov (United States)

    Cardiff, Robert D; Kenney, Nicholas

    2007-01-01

    For over a century, mouse mammary tumor biology and the associated Mouse mammary tumor virus (MMTV) have served as the foundation for experimental cancer research, in general, and, in particular, experimental breast cancer research. Spontaneous mouse mammary tumors were the basis for studies of the natural history of neoplasia, oncogenic viruses, host responses, endocrinology, and neoplastic progression. However, lacking formal proof of a human mammary tumor virus, the preeminence of the mouse model faded in the 1980s. Since the late 1980s, genetically engineered mice (GEM) have proven extremely useful for studying breast cancer and have become the animal model for human breast cancer. Hundreds of mouse models of human breast cancer have been developed since the first demonstration, in 1984, that the mouse mammary gland could be molecularly targeted and used to test the oncogenicity of candidate human genes. Now, very few scientists can avoid using a mouse model to test the biology of their favorite gene. The GEM have attracted a new generation of molecular and cellular biologists eager to apply their skills to these surrogates of the human disease. Newcomers often enter the field without an appreciation of the origins of mouse mammary tumor biology and the basis for many of the prevailing concepts. Our purpose in writing this short history of mouse mammary tumor biology is to provide a historical perspective for the benefit of the newcomers. If Einstein was correct in that "we stand on the shoulders of giants," the neophytes should meet their giants. PMID:17433908

  12. Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria.

    Directory of Open Access Journals (Sweden)

    Kyler B Pallister

    Full Text Available In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.

  13. Loss of the BRCA1-interacting helicase BRIP1 results in abnormal mammary acinar morphogenesis.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Daino

    Full Text Available BRIP1 is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1 and plays an important role in BRCA1-dependent DNA repair and DNA damage-induced checkpoint control. Recent studies implicate BRIP1 as a moderate/low-penetrance breast cancer susceptibility gene. However, the phenotypic effects of BRIP1 dysfunction and its role in breast cancer tumorigenesis remain unclear. To explore the function of BRIP1 in acinar morphogenesis of mammary epithelial cells, we generated BRIP1-knockdown MCF-10A cells by short hairpin RNA (shRNA-mediated RNA interference and examined its effect in a three-dimensional culture model. Genome-wide gene expression profiling by microarray and quantitative RT-PCR were performed to identify alterations in gene expression in BRIP1-knockdown cells compared with control cells. The microarray data were further investigated using the pathway analysis and Gene Set Enrichment Analysis (GSEA for pathway identification. BRIP1 knockdown in non-malignant MCF-10A mammary epithelial cells by RNA interference induced neoplastic-like changes such as abnormal cell adhesion, increased cell proliferation, large and irregular-shaped acini, invasive growth, and defective lumen formation. Differentially expressed genes, including MCAM, COL8A1, WIPF1, RICH2, PCSK5, GAS1, SATB1, and ELF3, in BRIP1-knockdown cells compared with control cells were categorized into several functional groups, such as cell adhesion, polarity, growth, signal transduction, and developmental process. Signaling-pathway analyses showed dysregulation of multiple cellular signaling pathways, involving LPA receptor, Myc, Wnt, PI3K, PTEN as well as DNA damage response, in BRIP1-knockdown cells. Loss of BRIP1 thus disrupts normal mammary morphogenesis and causes neoplastic-like changes, possibly via dysregulating multiple cellular signaling pathways functioning in the normal development of mammary glands.

  14. Absence of proteins related to murine mammary tumour virus polypeptides in rat mammary tumours

    International Nuclear Information System (INIS)

    Since normal rat DNA contains sequences which hybridize with the genome of the murine mammary tumour virus (MuMTV), it is possible that a related virus would play a role in mammary carcinogenesis in rats. The authors screened a number of rat mammary tumours for antigens related to the MuMTV polypeptides gp52 and p28 by means of a radioimmunoassay. (Auth.)

  15. Mammary gland tumors in captive African hedgehogs.

    Science.gov (United States)

    Raymond, J T; Gerner, M

    2000-04-01

    From December 1995 to July 1999, eight mammary gland tumors were diagnosed in eight adult captive female African hedgehogs (Atelerix albiventris). The tumors presented as single or multiple subcutaneous masses along the cranial or caudal abdomen that varied in size for each hedgehog. Histologically, seven of eight (88%) mammary gland tumors were malignant. Tumors were classified as solid (4 cases), tubular (2 cases), and papillary (2 cases). Seven tumors had infiltrated into the surrounding stroma and three tumors had histologic evidence of neoplastic vascular invasion. Three hedgehogs had concurrent neoplasms. These are believed to be the first reported cases of mammary gland tumors in African hedgehogs. PMID:10813628

  16. Evaluation of a topical herbal drug for its in-vivo immunomodulatory effect on cytokines production and antibacterial activity in bovine subclinical mastitis

    OpenAIRE

    Bhatt, Vaibhav D.; Tejas M Shah; Nauriyal, Dev S.; Kunjadia, Anju P.; Joshi, Chaitanya G.

    2014-01-01

    Background: Antibiotics have been in use in the treatment of bovine mastitis since decades; however, their use is associated with cost issues and human health concern. Use of herbal drugs does not generally carry these disadvantages. Many plants/herbs have been evaluated in the treatment of bovine mastitis with additional property of immunomodulation in affected mammary gland. Aim: To evaluate a topical herbal drug in two breeds of cattle for its in-vivo immunomodulatory effect on cytokines p...

  17. Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling.

    Science.gov (United States)

    Bagci, H; Laurin, M; Huber, J; Muller, W J; Côté, J-F

    2014-01-01

    Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also

  18. The use of liposomally-entrapped gentamicin in the treatment of bovine Staphylococcus aureus mastitis.

    OpenAIRE

    MacLeod, D L; Prescott, J F

    1988-01-01

    The effect of incorporation of gentamicin in liposomes on intracellular killing of Staphylococcus aureus was studied in vitro in cultured bovine mammary macrophages, and in experimental bovine mastitis. Liposomes were prepared by reverse-phase evaporation and ranged in size from 0.1 to 1.0 micron in diameter (mean 0.51 micron), with an encapsulation efficiency of gentamicin of 27.4%. Liposomes were taken up by in vitro cultured macrophages but intracellular killing of S. aureus over 12 h was ...

  19. Myoepithelial cells in canine mammary tumours.

    Science.gov (United States)

    Sánchez-Céspedes, Raquel; Millán, Yolanda; Guil-Luna, Silvia; Reymundo, Carlos; Espinosa de Los Monteros, Antonio; Martín de Las Mulas, Juana

    2016-01-01

    Mammary tumours are the most common neoplasms of female dogs. Compared to mammary tumours of humans and cats, myoepithelial (ME) cell involvement is common in canine mammary tumours (CMT) of any subtype. Since ME cell involvement in CMT influences both histogenetic tumour classification and prognosis, correct identification of ME cells is important. This review describes immunohistochemical methods for identification of canine mammary ME cells used in vivo. In addition, phenotypic and genotypic methods to isolate ME cells for in vitro studies to analyse tumour-suppressor protein production and gene expression are discussed. The contribution of ME cells to both histogenetic classifications and the prognosis of CMT is compared with other species and the potential use of ME cells as a method to identify carcinoma in situ is discussed. PMID:26639832

  20. Selenium in human mammary carcinogenesis

    DEFF Research Database (Denmark)

    Overvad, Kim; Grøn, P.; Langhoff, Otto;

    1991-01-01

    In a case-referent study on the possible role of selenium in human mammary carcinogenesis, serum selenium was found to be 79 +/- 12 micrograms/l in 66 cases and 81 +/- 12 micrograms/l in 93 referents. An internal trend in serum selenium was observed among cases (TNM stage I 81 +/- 11 micrograms....../l and TNM stage II 76 +/- 13 micrograms selenium/l), indicating disease-mediated changes. The evaluation of selenium as a risk indicator in human breast cancer was therefore restricted to TNM stage I patients (n = 36). Multiple logistic regression analyses including variables associated with selenium...... levels revealed no association between selenium levels and breast cancer risk....

  1. Caratterizzazione molecolare dei tumori mammari Tripli Negativi

    OpenAIRE

    Bertoni, Ramona

    2013-01-01

    I tumori mammari “Tripli Negativi” (TN) rappresentano circa il 15% di tutti i tumori mammari. Sono tumori clinicamente molto aggressivi, con una prognosi infausta: alta è l’incidenza di metastasi e di recidiva locale. Attualmente per pazienti affetti da questo tumore non esiste uno schema terapeutico efficace, né una terapia molecolare, infatti questi tumori non esprimono il recettore estrogenico (ER), quello progestinico (PGR) e il recettore HER2 (da cui il nome Tripli Negativi). Nella quasi...

  2. Mammary stem cells have myoepithelial cell properties

    OpenAIRE

    Prater, Michael D.; Petit, Val?rie; Russell, I Alasdair; Giraddi, Rajshekhar; Shehata, Mona; Menon, Suraj; Schulte, Reiner; Kalajzic, Ivo; Rath, Nicola; Olson, Michael F.; Metzger, Daniel; Faraldo, Marisa M.; Deugnier, Marie-Ange; Glukhova, Marina A.; Stingl, John

    2014-01-01

    Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepi...

  3. Expression of biologically active heterodimeric bovine follicle-stimulating hormone in milk of transgenic mice.

    OpenAIRE

    Greenberg, N M; Anderson, J.W.; Hsueh, A J; Nishimori, K; Reeves, J. J.; deAvila, D M; Ward, D N; Rosen, J. M.

    1991-01-01

    Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines o...

  4. An overview of bovine α-lactalbumin structure and functionality

    Directory of Open Access Journals (Sweden)

    Nicoleta STĂNCIUC

    2010-12-01

    Full Text Available α-Lactalbumin is the second major protein in bovine milk (2-5% of the total protein in bovine milk. The human variant has several physiologic functions in the neonatal period. In the mammary gland, itparticipates in lactose synthesis and facilitates milk production and secretion. α-Lactalbumin binds divalent cations (Ca2+, Zn2+ and may facilitate the absorption of essential minerals. Also, it provides awell-balanced supply of essential amino acids for the growi