Culture models of human mammary epithelial cell transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 overcomemore » 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.« less

Human Wharton's jelly stem cell conditioned medium and cell-free lysate inhibit human osteosarcoma and mammary carcinoma cell growth in vitro and in xenograft mice.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Arularasu, Suganya; Subramanian, Arjunan; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

2013-02-01

Human Wharton's jelly stem cells (hWJSCs) were shown to inhibit the growth of human mammary carcinomas. It is not known whether cell-free secretions or lysates of hWJSCs do the same on different cancers. They may be less controversial than cells to regulatory bodies for clinical application. We examined the influence of hWJSC conditioned medium (hWJSC-CM) and cell-free lysate (hWJSC-CL) on two osteosarcoma cell lines (MG-63, SKES-1) in vitro and on human mammary carcinomas in immunodeficient mice. When exposed to hWJSC-CL, increased vacuolations in MG-63 and increased membrane fragmentation in SKES-1 cells were observed, with greater cell death in SKES-1. Exposure of SKES-1 and MG-63 cells to hWJSC-CL showed significant decreases in cell proliferation of 46.48 ± 6.66% and 24.32 ± 5.67% respectively compared to controls. MG-63 and SKES-1 cells were annexin V-FITC positive and SKES-1 TUNEL positive following treatment with hWJSC-CM and hWJSC-CL. MG-63 cells were positive and SKES-1 cells negative for anti-BECLIN-1 and anti-LC3B following treatment with hWJSC-CM and hWJSC-CL. RT-PCR showed that the pro-apoptotic BAX gene and the autophagy-related ATG-5 and BECLIN-1 genes were up-regulated while the anti-apoptotic BCL2 and SURVIVIN genes were down-regulated in MG-63 and SKES-1 cells treated with hWJSC-CM and hWJSC-CL. Injections of hWJSCs and hWJSC-CM into mammary carcinomas in immunodeficient mice resulted in decreased tumor sizes and weights of 24.86 ± 6.05% to 37.03 ± 5.91% and 47.14 ± 7.36% to 55.09 ± 5.87% respectively at 6 weeks compared to controls. hWJSC-CM and hWJSC-CL inhibit mammary carcinoma and osteosarcoma cells via apoptosis and autophagy. Copyright © 2012 Wiley Periodicals, Inc.

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

PubMed Central

Band, V; Dalal, S; Delmolino, L; Androphy, E J

1993-01-01

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo. Images PMID:8387914

Radiation-induced chromosomal instability in human mammary epithelial cells

NASA Technical Reports Server (NTRS)

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.

Radiation-induced chromosomal instability in human mammary epithelial cells

NASA Astrophysics Data System (ADS)

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

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.

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

PubMed Central

CRUZ, PAMELA; TORRES, CRISTIAN; RAMÍREZ, MARÍA EUGENIA; EPUÑÁN, MARÍA JOSÉ; VALLADARES, LUIS EMILIO; SIERRALTA, WALTER DANIEL

2010-01-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E2) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E2, and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E2 in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E2-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels. PMID:22993572

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol.

PubMed

Cruz, Pamela; Torres, Cristian; Ramírez, María Eugenia; Epuñán, María José; Valladares, Luis Emilio; Sierralta, Walter Daniel

2010-05-01

The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E(2)) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E(2), and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E(2) in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E(2)-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels.

Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

PubMed Central

Ligueros, M.; Jeoung, D.; Tang, B.; Hochhauser, D.; Reidenberg, M. M.; Sonenberg, M.

1997-01-01

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9218727

Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells1

PubMed Central

Rowling, Matthew J.; Kemmis, Carly M.; Taffany, David A.; Welsh, JoEllen

2007-01-01

The major circulating form of vitamin D is 25-hydroxycholecalciferol [25(OH)D3], which is delivered to target tissues in complex with the serum vitamin D binding protein (DBP). We recently observed that mammary cells can metabolize 25(OH)D3 to 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the vitamin D receptor (VDR) ligand, and the objective of our study was to elucidate the mechanisms by which the 25(OH)D3-DBP complex is internalized by mammary cells prior to metabolism. Using fluorescent microscopy and temperature-shift techniques, we found that T-47D breast cancer cells rapidly internalize DBP via endocytosis, which is blunted by receptor-associated protein, a specific inhibitor of megalin-mediated endocytosis. Endocytosis of DBP was associated with activation of VDR by 25(OH)D3 but not 1,25(OH)2D3 (as measured by induction of the VDR target gene, CYP24). We also found that megalin and its endocytic partner, cubilin, are coexpressed in normal murine mammary tissue, in nontransformed human mammary epithelial cell lines, and in some established human breast cancer cell lines. To our knowledge, our studies are the first to demonstrate that mammary-derived cells express megalin and cubilin, which contribute to the endocytic uptake of 25(OH)D3-DBP and activation of the VDR pathway. PMID:17056796

An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

PubMed

Linnemann, Jelena R; Meixner, Lisa K; Miura, Haruko; Scheel, Christina H

2017-01-01

We have developed a three-dimensional organotypic culture system for primary human mammary epithelial cells (HMECs) in which the cells are cultured in free floating collagen type I gels. In this assay, luminal cells predominantly form multicellular spheres, while basal/myoepithelial cells form complex branched structures resembling terminal ductal lobular units (TDLUs), the functional units of the human mammary gland in situ. The TDLU-like organoids can be cultured for at least 3 weeks and can then be passaged multiple times. Subsequently, collagen gels can be stained with carmine or by immunofluorescence to allow for the analysis of morphology, protein expression and polarization, and to facilitate quantification of structures. In addition, structures can be isolated for gene expression analysis. In summary, this technique is suitable for studying branching morphogenesis, regeneration, and differentiation of HMECs as well as their dependence on the physical environment.

Hormesis effect of trace metals on cultured normal and immortal human mammary cells.

PubMed

Schmidt, Craig M; Cheng, Chun N; Marino, Angelo; Konsoula, Roula; Barile, Frank A

2004-06-01

An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDA-MB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green, and Celltiter blue fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine

Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2010-02-01

for mammary stem cells and be a target for transformation that results in the formation of aggressive mammary tumors. Breast cancer stem cells, Wnt...tumorigenesis, and human breast cancer. In addition, increasing evidence suggests that tumors arise from either normal stem or progenitor cells...population of mammary tumor cells that are CD24+/CD49++. Since Wnt pathway activation occurs in human breast cancer and is required for

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

PubMed Central

Teoh-Fitzgerald, ML; Fitzgerald, MP; Zhong, W; Askeland, RW; Domann, FE

2013-01-01

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging

Epigenetic regulation of normal human mammary cell type-specific miRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

Enhanced growth medium and method for culturing human mammary epithelial cells

DOEpatents

Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

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.

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

PubMed

Pellacani, Davide; Bilenky, Misha; Kannan, Nagarajan; Heravi-Moussavi, Alireza; Knapp, David J H F; Gakkhar, Sitanshu; Moksa, Michelle; Carles, Annaick; Moore, Richard; Mungall, Andrew J; Marra, Marco A; Jones, Steven J M; Aparicio, Samuel; Hirst, Martin; Eaves, Connie J

2016-11-15

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo.

PubMed

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis; Fre, Silvia

2013-10-14

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

PubMed Central

Lafkas, Daniel; Rodilla, Veronica; Huyghe, Mathilde; Mourao, Larissa; Kiaris, Hippokratis

2013-01-01

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells. PMID:24100291

Mammary Adipose Tissue-derived Lysophospholipids Promote Estrogen Receptor-negative Mammary Epithelial Cell Proliferation

PubMed Central

Volden, Paul A.; Skor, Maxwell N.; Johnson, Marianna B.; Singh, Puneet; Patel, Feenalie N.; McClintock, Martha K.; Brady, Matthew J.; Conzen, Suzanne D.

2016-01-01

Lysophosphatidic acid (LPA), acting in an autocrine or paracrine fashion through G protein-coupled receptors, has been implicated in many physiological and pathological processes including cancer. LPA is converted to lysophosphatidylcholine (LPC) by the secreted phospholipase, autotaxin (ATX). Although various cell types can produce ATX, adipocyte-derived ATX is believed to be the major source of circulating ATX and also to be the major regulator of plasma LPA. In addition to ATX, adipocytes secrete numerous other factors (adipokines); although several adipokines have been implicated in breast cancer biology, the contribution of mammary adipose tissue-derived LPC/ATX/LPA (LPA-axis) signaling to breast cancer is poorly understood. Using mammary fat-conditioned medium, we investigated the contribution of LPA signaling to mammary epithelial cancer cell biology and identified LPA signaling as a significant contributor to the oncogenic effects of the mammary adipose tissue secretome. To interrogate the role of mammary fat in the LPA-axis during breast cancer progression, we exposed mammary adipose tissue to secreted factors from estrogen receptor-negative mammary epithelial cell lines and monitored changes in the mammary fat pad LPA-axis. Our data indicate that bidirectional interactions between mammary cancer cells and mammary adipocytes alter the local LPA-axis and increase ATX expression in the mammary fat pad during breast cancer progression. Thus, the LPC/ATX/LPA axis may be a useful target for prevention in patients at risk of ER-negative breast cancer. PMID:26862086

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

NASA Technical Reports Server (NTRS)

Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

2000-01-01

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

USDA-ARS?s Scientific Manuscript database

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our objective was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretor...

Mammary stem cells and the differentiation hierarchy: current status and perspectives

PubMed Central

Visvader, Jane E.; Stingl, John

2014-01-01

The mammary epithelium is highly responsive to local and systemic signals, which orchestrate morphogenesis of the ductal tree during puberty and pregnancy. Based on transplantation and lineage tracing studies, a hierarchy of stem and progenitor cells has been shown to exist among the mammary epithelium. Lineage tracing has highlighted the existence of bipotent mammary stem cells (MaSCs) in situ as well as long-lived unipotent cells that drive morphogenesis and homeostasis of the ductal tree. Moreover, there is accumulating evidence for a heterogeneous MaSC compartment comprising fetal MaSCs, slow-cycling cells, and both long-term and short-term repopulating cells. In parallel, diverse luminal progenitor subtypes have been identified in mouse and human mammary tissue. Elucidation of the normal cellular hierarchy is an important step toward understanding the “cells of origin” and molecular perturbations that drive breast cancer. PMID:24888586

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

NASA Technical Reports Server (NTRS)

Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

1995-01-01

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells.

PubMed

Durante, M; Grossi, G F; Gialanella, G; Pugliese, M; Nappo, M; Yang, T C

1995-08-01

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS)

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

PubMed

McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Canonical Wnt Signaling as a Specific Mark of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2011-02-01

aggressive mammary tumors. 15. SUBJECT TERMS Breast cancer stem cells, Wnt signaling, canonical Wnt signaling, B-catenin, normal stem cells, adult stem...Wnt pathway is associated with abnormal mouse mammary development, tumorigenesis, and human breast cancer. In addition, increasing evidence suggests...activation occurs in human breast cancer and is required for proliferation of various other stem cell compartments, addressing how Wnt signaling promotes

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

PubMed Central

Zhao, Xiangshan; Malhotra, Gautam K.; Band, Hamid; Band, Vimla

2011-01-01

Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers. PMID:22279424

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes.

PubMed

Zhao, Xiangshan; Malhotra, Gautam K; Band, Hamid; Band, Vimla

2011-01-01

Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Mammary stem cells have myoepithelial cell properties

PubMed Central

Prater, Michael D.; Petit, Valérie; 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 myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy. PMID:25173976

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

PubMed

Casey, Alison E; Sinha, Ankit; Singhania, Rajat; Livingstone, Julie; Waterhouse, Paul; Tharmapalan, Pirashaanthy; Cruickshank, Jennifer; Shehata, Mona; Drysdale, Erik; Fang, Hui; Kim, Hyeyeon; Isserlin, Ruth; Bailey, Swneke; Medina, Tiago; Deblois, Genevieve; Shiah, Yu-Jia; Barsyte-Lovejoy, Dalia; Hofer, Stefan; Bader, Gary; Lupien, Mathieu; Arrowsmith, Cheryl; Knapp, Stefan; De Carvalho, Daniel; Berman, Hal; Boutros, Paul C; Kislinger, Thomas; Khokha, Rama

2018-06-19

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology. © 2018 Casey et al.

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

PubMed Central

Rosenfield, Sonia M.; Bowden, Emma T.; Cohen-Missner, Shani; Gibby, Krissa A.; Ory, Virginie; Henke, Ralf T.; Riegel, Anna T.; Wellstein, Anton

2012-01-01

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development. PMID:23077670

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

PubMed

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

PubMed

Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

2018-06-01

The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer, arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5 + cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5 + cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. © 2018 Sun et al.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

PubMed Central

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells12

PubMed Central

Chlon, Timothy M.; Taffany, David A.; Welsh, JoEllen; Rowling, Matthew J.

2008-01-01

The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells. PMID:18567755

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

PubMed Central

1993-01-01

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes.

PubMed

Bazarov, Alexey V; Hines, William C; Mukhopadhyay, Rituparna; Beliveau, Alain; Melodyev, Sonya; Zaslavsky, Yuri; Yaswen, Paul

2009-10-15

A central question in breast cancer biology is how cancer cells acquire telomerase activity required for unlimited proliferation. According to one model, proliferation of telomerase(-) pre-malignant cells leads to telomere dysfunction and increased genomic instability. Such instability leads in rare cases to reactivation of telomerase and immortalization. The mechanism of telomerase reactivation remains unknown. We have studied immortalization of cultured human mammary epithelial cells by c-Myc, a positive transcriptional regulator of the hTERT gene encoding the catalytic subunit of telomerase. Retrovirally introduced c-Myc cDNA resulted in immortalization of human mammary epithelial cells in which the cyclin dependent kinase inhibitor, p16(INK4A), was inactivated by an shRNA-encoding retrovirus. However, while c-Myc introduction immediately resulted in increased activity of transiently transfected hTERT promoter reporter constructs, endogenous hTERT mRNA levels did not change until about 60 population doublings after c-Myc introduction. Increased endogenous hTERT transcripts and stabilization of telomeric DNA in cells expressing exogenous c-Myc coincided with telomere dysfunction-associated senescence in control cultures. Genome copy number analyses of immortalized cells indicated amplifications of some or all of chromosome 5, where hTERT genes are located. hTERT gene copy number, however, was not increased in one case. The results are consistent with the hypothesis that changes in chromosome 5, while not necessarily increasing hTERT gene copy number, resulted in removal of repressive chromatin structures around hTERT loci, allowing induction of hTERT transcription. These in vitro results model one possible sequence of events leading to immortalization of breast epithelial cells during cancer progression.

Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours.

PubMed

Nathrath, W B; Wilson, P D; Trejdosiewicz, L K

1982-01-01

Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.

Genes involved in immortalization of human mammary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stampfer, Martha R.; Yaswen, Paul

2001-09-27

Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings ofmore » this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of

Dicer in Mammary Tumor Stem Cell Maintenance

DTIC Science & Technology

2006-03-01

we are cloning small RNAs from mammary stem cells in order to determine the regulatory niches that miRNAs may fill in this cell type. Our ultimate goal is to assess the role of Dicer in mammary tumor stem cell maintenance.

The effect of neighboring cells on the stiffness of cancerous and non-cancerous human mammary epithelial cells

NASA Astrophysics Data System (ADS)

Guo, Xinyi; Bonin, Keith; Scarpinato, Karin; Guthold, Martin

2014-10-01

Using an Atomic Force Microscope (AFM) with a 5.3 μm diameter spherical probe, we determined mechanical properties of individual human mammary epithelial cells. The cells were derived from a pair of cell lines that mimic cell progression through four phases of neoplastic transformation: normal (non-transformed), immortal, tumorigenic, and metastatic. Measurements on cells in all four phases were taken over both the cytoplasmic and nuclear regions. Moreover, the measurements were made for cells in different microenvironments as related to cell-cell contacts: isolated cells; cells residing on the periphery of a contiguous cell monolayer; and cells on the inside of a contiguous cell monolayer. By fitting the AFM force versus indentation curves to a Hertz model, we determined the pseudo-elastic Young’s modulus, E. Combining all data for the cellular subregions (over nucleus and cytoplasm) and the different cell microenvironments, we obtained stiffness values for normal, immortal, tumorigenic, and metastatic cells of 870 Pa, 870 Pa, 490 Pa, and 580 Pa, respectively. That is, cells become softer as they advance to the tumorigenic phase and then stiffen somewhat in the final step to metastatic cells. We also found a distinct contrast in the influence of a cell’s microenvironment on cell stiffness. Normal mammary epithelial cells inside a monolayer are stiffer than peripheral cells, which are stiffer than isolated cells. However, the microenvironment had a slight, opposite effect on tumorigenic and little effect on immortal and metastatic cell stiffness. Thus, the stiffness of cancer cells is less sensitive to the microenvironment than normal cells. Our results show that the mechanical properties of a cell can depend on cancer progression and microenvironment (cell-cell interactions).

Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

PubMed

Fu, Mei; Chen, Yabing; Xiong, Xianrong; Lan, Daoliang; Li, Jian

2014-01-01

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

Bovine mammary stem cells: Cell biology meets production agriculture

USDA-ARS?s Scientific Manuscript database

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 ...

Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer.

PubMed

Malik, Durr-E-Shahwar; David, Rhiannon M; Gooderham, Nigel J

2018-04-01

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10 -7 -10 -4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10 -3 -10 -1 M) with PhIP (10 -7 -10 -4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

PubMed

Bird, R Curtis; Deinnocentes, Patricia; Church Bird, Allison E; van Ginkel, Frederik W; Lindquist, Joni; Smith, Bruce F

2011-01-01

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

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

NASA Technical Reports Server (NTRS)

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.

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

PubMed

Riches, Andrew; Campbell, Elaine; Borger, Eva; Powis, Simon

2014-03-01

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microenvironmental Regulation of Mammary Carcinogenesis

DTIC Science & Technology

2008-06-01

cells. These models share many of the hallmarks of multistage human breast cancer development including histological disease progression and immune cell... developed by Muller and colleagues20, represents a reasonable recapitulation of late-stage human breast cancer as determined by histological progression ...Annual Progress Report d. Develop a profile of proteolytic activities in normal and neoplastic mammary tissues from mouse models of mammary

SOCS3 promotes apoptosis of mammary differentiated cells.

PubMed

Le Provost, Fabienne; Miyoshi, Keiko; Vilotte, Jean-Luc; Bierie, Brian; Robinson, Gertraud W; Hennighausen, Lothar

2005-12-30

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).

Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition

PubMed Central

McCoy, Erica L.; Iwanaga, Ritsuko; Jedlicka, Paul; Abbey, Nee-Shamo; Chodosh, Lewis A.; Heichman, Karen A.; Welm, Alana L.; Ford, Heide L.

2009-01-01

Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell–like features. PMID:19726883

Sequencing the transcriptome of milk production: milk trumps mammary tissue

PubMed Central

2013-01-01

Background Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation. PMID:24330573

Sequencing the transcriptome of milk production: milk trumps mammary tissue.

PubMed

Lemay, Danielle G; Hovey, Russell C; Hartono, Stella R; Hinde, Katie; Smilowitz, Jennifer T; Ventimiglia, Frank; Schmidt, Kimberli A; Lee, Joyce W S; Islas-Trejo, Alma; Silva, Pedro Ivo; Korf, Ian; Medrano, Juan F; Barry, Peter A; German, J Bruce

2013-12-12

Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wazer, D.E.; Chu, Qiuming; Liu, Xiao Long

1994-04-01

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated [gamma]-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletionmore » within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G[sub 1] arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells. 44 refs., 8 figs., 1 tab.« less

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booth, Brian W., E-mail: brbooth@clemson.edu; Institute for Biological Interfaces of Engineering, Clemson University, Clemson, SC 29634; Boulanger, Corinne A.

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signalingmore » pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.« less

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics.

PubMed

Booth, Brian W; Boulanger, Corinne A; Anderson, Lisa H; Jimenez-Rojo, Lucia; Brisken, Cathrin; Smith, Gilbert H

2010-02-01

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D beta-geo (CDbetageo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDbetageo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG(-/-) mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro. Copyright 2009 Elsevier Inc. All rights reserved.

Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

2016-04-22

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells. Copyright © 2016 Elsevier Inc. All rights reserved.

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

NASA Technical Reports Server (NTRS)

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).

Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours

PubMed Central

Messal, Hendrik A.; Andersson, Agneta B.; Ruiz, E. Josue; Gerling, Marco; Douagi, Iyadh; Spencer-Dene, Bradley; Musch, Alexandra; Mitter, Richard; Bhaw, Leena; Stone, Richard; Bornhorst, Dorothee; Sesay, Abdul K.; Jonkers, Jos; Stamp, Gordon; Malanchi, Ilaria; Toftgård, Rune; Behrens, Axel

2018-01-01

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or upon stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumourigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance. PMID:27798604

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

PubMed Central

Chanson, Lea; Brownfield, Douglas; Garbe, James C.; Kuhn, Irene; Stampfer, Martha R.; Bissell, Mina J.; LaBarge, Mark A.

2011-01-01

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. PMID:21300877

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

PubMed

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 (P<0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. © 2014 Japanese Society of Animal Science.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells.

PubMed

Wen, G; Pachner, L I; Gessner, D K; Eder, K; Ringseis, R

2016-11-01

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the

Mechanical strain induces involution-associated events in mammary epithelial cells

PubMed Central

Quaglino, Ana; Salierno, Marcelo; Pellegrotti, Jesica; Rubinstein, Natalia; Kordon, Edith C

2009-01-01

Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device

Synergistic effect of sequential administration of mitoguazone (MGBG) and gemcitabine in treating tissue cultured human breast cancer cells and mammary rat tumors.

PubMed

Ishmael, D Richard; Chen, Wei R; Nordquist, John A; Liu, Hong; Nordquist, Robert E

2003-04-01

Modulation of cancer chemotherapeutic drugs has been attempted to increase efficacy and overcome resistance to the chemotherapeutic agent. Studies have shown schedule-dependent interactions in combined use of chemotherapeutic drugs. Mitoguazone (MGBG), an old drug with possible modulating activity, was used in combination with gemcitabine, a relatively new cancer drug, in treating tissue cultured human breast cancer cells and mammary rat tumors. Tissue cultured BOT-2 cancer cells were first treated with varying concentrations of gemcitabine and MGBG, independently. Combinations of the two drugs were then used with different scheduled administrations. Marked synergistic activity was found between gemcitabine and MGBG when the MGBG was given first, followed by gemcitabine 24 hours later. A non-toxic dose of MGBG enhanced the toxicity of gemcitabine by eight orders of magnitude using MTT assays in the tissue cultured human breast cancer cell study. The sequential administration of MGBG and gemcitabine also increased the survival rate of rats bearing mammary tumors in our pilot animal study.

The Origin and Significance of Mammary Intraductal Foam Cells

DTIC Science & Technology

2005-09-01

hematopoeitic origin developed in mammary tissue with both benign and malignant differentiation, depending on environmental cues. Progression of the cells...contribution of hematopoeitic precursors to the heterogeneity of cell types in benign and malignant mammary tissue.

Characterization of mammary epithelial stem/progenitor cells and their changes with aging in common marmosets.

PubMed

Wu, Anqi; Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Chen, Yuanhong; Zhang, Fuchuang; Bandyopadhyay, Abhik; Wang, Danhan; Gorena, Karla M; Huang, Changjiang; Tardif, Suzette; Nathanielsz, Peter W; Sun, Lu-Zhe

2016-08-25

Age is the number one risk factor for breast cancer, yet the underlying mechanisms are unexplored. Age-associated mammary stem cell (MaSC) dysfunction is thought to play an important role in breast cancer carcinogenesis. Non-human primates with their close phylogenetic relationship to humans provide a powerful model system to study the effects of aging on human MaSC. In particular, the common marmoset monkey (Callithrix jacchus) with a relatively short life span is an ideal model for aging research. In the present study, we characterized for the first time the mammary epithelial stem/progenitor cells in the common marmoset. The MaSC-enriched cells formed four major types of morphologically distinct colonies when cultured on plates pre-seeded with irradiated NIH3T3 fibroblasts, and were also capable of forming mammospheres in suspension culture and subsequent formation of 3D organoids in Matrigel culture. Most importantly, these 3D organoids were found to contain stem/progenitor cells that can undergo self-renewal and multi-lineage differentiation both in vitro and in vivo. We also observed a significant decrease of luminal-restricted progenitors with age. Our findings demonstrate that common marmoset mammary stem/progenitor cells can be isolated and quantified with established in vitro and in vivo assays used for mouse and human studies.

Apigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells.

PubMed

Coombs, Melanie R Power; Harrison, Megan E; Hoskin, David W

2016-10-01

Programmed death ligand 1 (PD-L1) is expressed by many cancer cell types, as well as by activated T cells and antigen-presenting cells. Constitutive and inducible PD-L1 expression contributes to immune evasion by breast cancer (BC) cells. We show here that the dietary phytochemical apigenin inhibited interferon (IFN)-γ-induced PD-L1 upregulation by triple-negative MDA-MB-468 BC cells, HER2(+) SK-BR-3 BC cells, and 4T1 mouse mammary carcinoma cells, as well as human mammary epithelial cells, but did not affect constitutive PD-L1 expression by triple-negative MDA-MB-231 BC cells. IFN-β-induced expression of PD-L1 by MDA-MB-468 cells was also inhibited by apigenin. In addition, luteolin, the major metabolite of apigenin, inhibited IFN-γ-induced PD-L1 expression by MDA-MB-468 cells. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 and 4T1 cells was associated with reduced phosphorylation of STAT1, which was early and transient at Tyr701 and sustained at Ser727. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 cells also increased proliferation and interleukin-2 synthesis by PD-1-expressing Jurkat T cells that were co-cultured with MDA-MB-468 cells. Apigenin therefore has the potential to increase the vulnerability of BC cells to T cell-mediated anti-tumor immune responses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos.

PubMed

Gui, Tao; Liu, Xing; Tao, Jia; Chen, Jianwen; Li, Yunsheng; Zhang, Meiling; Wu, Ronghua; Zhang, Yuanliang; Peng, Kaisong; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2013-12-01

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland. Copyright © 2013 Elsevier B.V. All rights reserved.

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

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

PubMed

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.

STAT signaling in mammary gland differentiation, cell survival and tumorigenesis.

PubMed

Haricharan, S; Li, Y

2014-01-25

The mammary gland is a unique organ that undergoes extensive and profound changes during puberty, menstruation, pregnancy, lactation and involution. The changes that take place during puberty involve large-scale proliferation and invasion of the fat-pad. During pregnancy and lactation, the mammary cells are exposed to signaling pathways that inhibit apoptosis, induce proliferation and invoke terminal differentiation. Finally, during involution the mammary gland is exposed to milk stasis, programmed cell death and stromal reorganization to clear the differentiated milk-producing cells. Not surprisingly, the signaling pathways responsible for bringing about these changes in breast cells are often subverted during the process of tumorigenesis. The STAT family of proteins is involved in every stage of mammary gland development, and is also frequently implicated in breast tumorigenesis. While the roles of STAT3 and STAT5 during mammary gland development and tumorigenesis are well studied, others members, e.g. STAT1 and STAT6, have only recently been observed to play a role in mammary gland biology. Continued investigation into the STAT protein network in the mammary gland will likely yield new biomarkers and risk factors for breast cancer, and may also lead to novel prophylactic or therapeutic strategies against breast cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

STAT signaling in mammary gland differentiation, cell survival and tumorigenesis

PubMed Central

Haricharan, S; Li, Y

2013-01-01

The mammary gland is a unique organ that undergoes extensive and profound changes during puberty, menstruation, pregnancy, lactation and involution. The changes that take place during puberty involve large-scale proliferation and invasion of the fat-pad. During pregnancy and lactation, the mammary cells are exposed to signaling pathways that inhibit apoptosis, induce proliferation and invoke terminal differentiation. Finally, during involution the mammary gland is exposed to milk stasis, programed cell death and stromal reorganization to clear the differentiated milk-producing cells. Not surprisingly, the signaling pathways responsible for bringing about these changes in breast cells are often subverted during the process of tumorigenesis. The STAT family of proteins is involved in every stage of mammary gland development, and is also frequently implicated in breast tumorigenesis. While the roles of STAT3 and STAT5 during mammary gland development and tumorigenesis are well studied, others members, e.g. STAT1 and STAT6, have only recently been observed to play a role in mammary gland biology. Continued investigation into the STAT protein network in the mammary gland will likely yield new biomarkers and risk factors for breast cancer, and may also lead to novel prophylactic or therapeutic strategies against breast cancer. PMID:23541951

hEcd, A Novel Regulator of Mammary Epithelial Cell Survival

DTIC Science & Technology

2009-09-01

theYeast Two hybrid analysis with human papilloma virus oncogene E6 (the most efficient oncogene to immortalize hMECs in vitro) as a bait and mammary...transformation. We have identified a novel protein us ing the Yeast Two hybrid analysis with human papilloma virus oncogene E6 (the most efficient...epithelial cell cDNA library, we identified hEcd ( human orthologue of Drosophila Ecdysoneless) as a novel E6 binding partner. To study the cellular

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

NASA Technical Reports Server (NTRS)

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

2001-01-01

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ewan, Kenneth B.R.; Oketch-Rabah, Hellen A.; Ravani, Shraddha A.

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}more » 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.« less

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

PubMed

Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

2009-06-01

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells

DOE PAGES

Sridharan, Deepa M.; Enerio, Shiena; Stampfer, Martha M.; ...

2017-02-28

Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations andmore » changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.« less

Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sridharan, Deepa M.; Enerio, Shiena; Stampfer, Martha M.

Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations andmore » changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.« less

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

PubMed Central

Guo, Qiuchen; Burchard, Julja; Spellman, Paul

2017-01-01

Women diagnosed with breast cancer within 5 years of childbirth have poorer prognosis than nulliparous or pregnant women. Weaning-induced breast involution is implicated, as the collagen-rich, immunosuppressive microenvironment of the involuting mammary gland is tumor promotional in mice. To investigate the role of mammary fibroblasts, isolated mammary PDGFRα+ cells from nulliparous and postweaning mice were assessed for activation phenotype and protumorigenic function. Fibroblast activation during involution was evident by increased expression of fibrillar collagens, lysyl oxidase, Tgfb1, and Cxcl12 genes. The ability of mammary tumors to grow in an isogenic, orthotopic transplant model was increased when tumor cells were coinjected with involution-derived compared with nulliparous-derived mammary fibroblasts. Mammary tumors in the involution-fibroblast group had increased Ly6C+ monocytes at the tumor border, and decreased CD8+ T cell infiltration and tumor cell death. Ibuprofen treatment suppressed involution-fibroblast activation and tumor promotional capacity, concurrent with decreases in tumor Ly6C+ monocytes, and increases in intratumoral CD8+ T cell infiltration, granzyme levels, and tumor cell death. In total, our data identify a COX/prostaglandin E2 (PGE2)–dependent activated mammary fibroblast within the involuting mammary gland that displays protumorigenic, immunosuppressive activity, identifying fibroblasts as potential targets for the prevention and treatment of postpartum breast cancer. PMID:28352652

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

In vitro expansion of the mammary stem/progenitor cell population by xanthosinetreatment

USDA-ARS?s Scientific Manuscript database

Background: Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine (Xs) treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo ...

Mammary Stem Cells: Premise, Properties, and Perspectives.

PubMed

Lloyd-Lewis, Bethan; Harris, Olivia B; Watson, Christine J; Davis, Felicity M

2017-08-01

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

PubMed Central

Tenan, Mirna; Ferrari, Paolo; Sappino, André‐Pascal

2016-01-01

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl3)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens. PMID:27541736

Metastatic canine mammary carcinomas can be identified by a gene expression profile that partly overlaps with human breast cancer profiles

PubMed Central

2010-01-01

Background Similar to human breast cancer mammary tumors of the female dog are commonly associated with a fatal outcome due to the development of distant metastases. However, the molecular defects leading to metastasis are largely unknown and the value of canine mammary carcinoma as a model for human breast cancer is unclear. In this study, we analyzed the gene expression signatures associated with mammary tumor metastasis and asked for parallels with the human equivalent. Methods Messenger RNA expression profiles of twenty-seven lymph node metastasis positive or negative canine mammary carcinomas were established by microarray analysis. Differentially expressed genes were functionally characterized and associated with molecular pathways. The findings were also correlated with published data on human breast cancer. Results Metastatic canine mammary carcinomas had 1,011 significantly differentially expressed genes when compared to non-metastatic carcinomas. Metastatic carcinomas had a significant up-regulation of genes associated with cell cycle regulation, matrix modulation, protein folding and proteasomal degradation whereas cell differentiation genes, growth factor pathway genes and regulators of actin organization were significantly down-regulated. Interestingly, 265 of the 1,011 differentially expressed canine genes are also related to human breast cancer and, vice versa, parts of a human prognostic gene signature were identified in the expression profiles of the metastatic canine tumors. Conclusions Metastatic canine mammary carcinomas can be discriminated from non-metastatic carcinomas by their gene expression profiles. More than one third of the differentially expressed genes are also described of relevance for human breast cancer. Many of the differentially expressed genes are linked to functions and pathways which appear to be relevant for the induction and maintenance of metastatic progression and may represent new therapeutic targets. Furthermore, dogs

Mouse mammary tumour virus (MMTV) and human breast cancer with neuroendocrine differentiation.

PubMed

Js, Lawson; Cc, Ngan; Wk, Glenn; Dd, Tran

2017-01-01

Mouse mammary tumour viruses (MMTVs) may have a role in a subset of human breast cancers. MMTV positive human breast cancers have similar histological characteristics to neuroendocrine breast cancers and to MMTV positive mouse mammary tumours. The purpose of this study was to investigate the expression of neuroendocrine biomarkers - synaptophysin and chromogranin, to determine if these histological characteristics and biomarker expression were due to the influences of MMTV. Immunohistochemistry analyses to identify synaptophysin and chromogranin were conducted on a series of human breast cancers in which (i) MMTV had been previously identified and had similar histological characteristics to MMTV positive mouse mammary tumours and (ii) MMTV positive mouse mammary tumours. The expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumors were all positive (7 of 7 specimens - 100% positive). The expression of synaptophysin and chromogranin in MMTV positive human breast cancers was much less prevalent (3 of 22 - 14%). There was no expression of synaptophysin and chromogranin in the normal breast tissue control specimens. It is not possible to draw any firm conclusions from these observations. However, despite the small numbers of MMTV positive mouse mammary tumours in this study, the universal expression in these specimens of synaptophysin and chromogranin proteins is striking. This pattern of synaptophysin and chromogranin expression is very different from their expression in MMTV positive human breast cancers. The reason for these differences is not known. The high prevalence of positive expression of synaptophysin and chromogranin in MMTV positive mouse mammary tumours and low expression of synaptophysin and chromogranin in MMTV positive human breast cancers indicates that MMTV is not usually associated with neuroendocrine human breast cancers.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chanson, L.; Brownfield, D.; Garbe, J. C.

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 humanmore » 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.« less

Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells

PubMed Central

Chamard-Jovenin, Clémence; Thiebaut, Charlène; Chesnel, Amand; Bresso, Emmanuel; Morel, Chloé; Smail-Tabbone, Malika; Devignes, Marie-Dominique; Boukhobza, Taha; Dumond, Hélène

2017-01-01

Fetal and neonatal exposure to long-chain alkylphenols has been suspected to promote breast developmental disorders and consequently to increase breast cancer risk. However, disease predisposition from developmental exposures remains unclear. In this work, human MCF-10A mammary epithelial cells were exposed in vitro to a low dose of a realistic (4-nonylphenol + 4-tert-octylphenol) mixture. Transcriptome and cell-phenotype analyses combined to functional and signaling network modeling indicated that long-chain alkylphenols triggered enhanced proliferation, migration ability, and apoptosis resistance and shed light on the underlying molecular mechanisms which involved the human estrogen receptor alpha 36 (ERα36) variant. A male mouse-inherited transgenerational model of exposure to three environmentally relevant doses of the alkylphenol mix was set up in order to determine whether and how it would impact on mammary gland architecture. Mammary glands from F3 progeny obtained after intrabuccal chronic exposure of C57BL/6J P0 pregnant mice followed by F1–F3 male inheritance displayed an altered histology which correlated with the phenotypes observed in vitro in human mammary epithelial cells. Since cellular phenotypes are similar in vivo and in vitro and involve the unique ERα36 human variant, such consequences of alkylphenol exposure could be extrapolated from mouse model to human. However, transient alkylphenol treatments combined to ERα36 overexpression in mammary epithelial cells were not sufficient to trigger tumorigenesis in xenografted Nude mice. Therefore, it remains to be determined if low-dose alkylphenol transgenerational exposure and subsequent abnormal mammary gland development could account for an increased breast cancer susceptibility. PMID:29109696

Human mammary epithelial cells exhibit a bimodal correlated random walk pattern.

PubMed

Potdar, Alka A; Jeon, Junhwan; Weaver, Alissa M; Quaranta, Vito; Cummings, Peter T

2010-03-10

Organisms, at scales ranging from unicellular to mammals, have been known to exhibit foraging behavior described by random walks whose segments confirm to Lévy or exponential distributions. For the first time, we present evidence that single cells (mammary epithelial cells) that exist in multi-cellular organisms (humans) follow a bimodal correlated random walk (BCRW). Cellular tracks of MCF-10A pBabe, neuN and neuT random migration on 2-D plastic substrates, analyzed using bimodal analysis, were found to reveal the BCRW pattern. We find two types of exponentially distributed correlated flights (corresponding to what we refer to as the directional and re-orientation phases) each having its own correlation between move step-lengths within flights. The exponential distribution of flight lengths was confirmed using different analysis methods (logarithmic binning with normalization, survival frequency plots and maximum likelihood estimation). Because of the presence of non-uniform turn angle distribution of move step-lengths within a flight and two different types of flights, we propose that the epithelial random walk is a BCRW comprising of two alternating modes with varying degree of correlations, rather than a simple persistent random walk. A BCRW model rather than a simple persistent random walk correctly matches the super-diffusivity in the cell migration paths as indicated by simulations based on the BCRW model.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

PubMed Central

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

The Analysis of Cell Population Dynamics in Mammary Gland Development and Tumorigenesis

DTIC Science & Technology

2005-08-01

AD Award Number: DAMD17-03-1-0498 TITLE: The Analysis of Cell Population Dynamics in Mammary Gland Development and Tumorigenesis PRINCIPAL...Summary 1 Aug 2004 - 31 Jul 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER The Analysis of Cell Population Dynamics in Mammary Gland Development and...STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The mammary gland is made up of several epithelial cell

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line

PubMed Central

Foreman, Jennifer E.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2009-01-01

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis. PMID:19660859

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

PubMed Central

Kerjaschki, Dontscho; Bago-Horvath, Zsuzsanna; Rudas, Margaretha; Sexl, Veronika; Schneckenleithner, Christine; Wolbank, Susanne; Bartel, Gregor; Krieger, Sigurd; Kalt, Romana; Hantusch, Brigitte; Keller, Thomas; Nagy-Bojarszky, Katalin; Huttary, Nicole; Raab, Ingrid; Lackner, Karin; Krautgasser, Katharina; Schachner, Helga; Kaserer, Klaus; Rezar, Sandra; Madlener, Sybille; Vonach, Caroline; Davidovits, Agnes; Nosaka, Hitonari; Hämmerle, Monika; Viola, Katharina; Dolznig, Helmut; Schreiber, Martin; Nader, Alexander; Mikulits, Wolfgang; Gnant, Michael; Hirakawa, Satoshi; Detmar, Michael; Alitalo, Kari; Nijman, Sebastian; Offner, Felix; Maier, Thorsten J.; Steinhilber, Dieter; Krupitza, Georg

2011-01-01

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas. PMID:21540548

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

PubMed Central

Pathania, Rajneesh; Ramachandran, Sabarish; Elangovan, Selvakumar; Padia, Ravi; Yang, Pengyi; Cinghu, Senthilkumar; Veeranan-Karmegam, Rajalakshmi; Arjunan, Pachiappan; Gnana-Prakasam, Jaya P.; Fulzele, Sadanand; Pei, Lirong; Chang, Chang-Sheng; Choi, Hyeon; Shi, Huidong; Manicassamy, Santhakumar; Prasad, Puttur D.; Sharma, Suash; Ganapathy, Vadivel; Jothi, Raja; Thangaraju, Muthusamy

2015-01-01

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumors, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumors and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment. PMID:25908435

DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis.

PubMed

Pathania, Rajneesh; Ramachandran, Sabarish; Elangovan, Selvakumar; Padia, Ravi; Yang, Pengyi; Cinghu, Senthilkumar; Veeranan-Karmegam, Rajalakshmi; Arjunan, Pachiappan; Gnana-Prakasam, Jaya P; Sadanand, Fulzele; Pei, Lirong; Chang, Chang-Sheng; Choi, Jeong-Hyeon; Shi, Huidong; Manicassamy, Santhakumar; Prasad, Puttur D; Sharma, Suash; Ganapathy, Vadivel; Jothi, Raja; Thangaraju, Muthusamy

2015-04-24

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

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

PubMed

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.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Methylation of p16(INK4a) promoters occurs in vivo in histologically normal human mammary epithelia

NASA Technical Reports Server (NTRS)

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.

An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

PubMed Central

Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. Steven; Qian, Wei-Jun

2010-01-01

Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

PKCθ promotes c-Rel–driven mammary tumorigenesis in mice and humans by repressing estrogen receptor α synthesis

PubMed Central

Belguise, Karine; Sonenshein, Gail E.

2007-01-01

The vast majority of primary human breast cancer tissues display aberrant nuclear NF-κB c-Rel expression. A causal role for c-Rel in mammary tumorigenesis has been demonstrated using a c-Rel transgenic mouse model; however, tumors developed with a long latency, suggesting a second event is needed to trigger tumorigenesis. Here we show that c-Rel activity in the mammary gland is repressed by estrogen receptor α (ERα) signaling, and we identify an epigenetic mechanism in breast cancer mediated by activation of what we believe is a novel PKCθ-Akt pathway that leads to downregulation of ERα synthesis and derepression of c-Rel. ERα levels were lower in c-Rel–induced mammary tumors compared with normal mammary gland tissue. PKCθ induced c-Rel activity and target gene expression and promoted growth of c-Rel- and c-RelxCK2α–driven mouse mammary tumor–derived cell lines. RNA expression levels of PKCθ and c-Rel target genes were inversely correlated with ERα levels in human breast cancer specimens. PKCθ activated Akt, thereby inactivating forkhead box O protein 3a (FOXO3a) and leading to decreased synthesis of its target genes, ERα and p27Kip1. Thus we have shown that activation of PKCθ inhibits the FOXO3a/ERα/p27Kip1 axis that normally maintains an epithelial cell phenotype and induces c-Rel target genes, thereby promoting proliferation, survival, and more invasive breast cancer. PMID:18037997

Isolation of Estrogen Regulated Genes from MCF-7 Human Mammary Cancer Cells

DTIC Science & Technology

1990-07-13

Syrian hamster BHK cells. The expression of approximately five hundred clones increased at various points in Gl transit (Hirschhom er d., 1984). No...hyperplasia of ductal epithelium of the mammary gland (Lyons etal., 1958). Biochemical changes in uterine wet weight, protein content, and nucleic... transition from a resting to a growing state (Kaufman and Sharp, 1983). Similarly post- transcriptional processing appears to be important in the ceU

5-Fluorouracil may enrich cancer stem cells in canine mammary tumor cells in vitro.

PubMed

Zhou, Bin; Jin, Yipeng; Zhang, Di; Lin, Degui

2018-05-01

Mammary gland carcinomas are the most common neoplasms in women and unsterilized female dogs. Owing to the existence of cancer stem cells (CSCs), chemotherapy is not able to cure these types of diseases completely. A number of studies have demonstrated that CSCs are resistant to chemotherapeutic drugs, but whether canine mammary tumor cells that have acquired resistance to 5-fluorouracil (5-FU) exhibited properties of CSCs remains unknown. The aim of the present study was to investigate whether 5-fluorouracil-resistant canine mammary tumor cells exhibited properties of CSCs. CSCs were analyzed using western blot assays, ultra-low attachment sphere cultures, flow cytometry and migration (wound healing and Transwell) assays. The results indicated that, compared with parental cells, proteins associated with the Wnt/β-catenin signaling pathway and aldehyde dehydrogenase 1 were overexpressed, the number and size of spheres in the 5-FU-resistant cells were increased, the ratio of CD44 + /CD24 -/low cells was increased and the migratory ability was improved in vitro compared with the 5-FU-susceptible cells. In conclusion, stimulation with chemotherapeutic drugs including 5-FU is a good method for increasing the proportion of canine mammary tumor stem cells in vitro , which may provide further understanding of chemotherapeutic methods and CSCs.

Comparison of the transcriptpmes of long-tern label retaining-cells and C cells microdissected from mammary epithelium: an initial study to character potential stem/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) account for the cell lineage of mammary epithelia and provide for mammary growth, development and tissue homeostasis. The presence of MaSC was clearly demonstrated by the generation of an entire mammary gland from a single cell implanted into epithelium-ablated mammary fat...

Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis.

PubMed

Greenwood, Erin; Wrenn, Emma D; Cheung, Kevin J

2017-02-27

The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of cloned embryos from caprine mammary epithelial cells expressing recombinant human β-defensin-3.

PubMed

Liu, Jun; Luo, Yan; Liu, Qingqing; Zheng, Liming; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-03-01

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway

PubMed Central

Voutilainen, Maria; Lönnblad, Darielle; Shirokova, Vera; Elo, Teresa; Rysti, Elisa; Schmidt-Ullrich, Ruth; Schneider, Pascal; Mikkola, Marja L.

2015-01-01

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants. PMID:26581094

Canine mammary tumors as a model for human disease.

PubMed

Abdelmegeed, Somaia M; Mohammed, Sulma

2018-06-01

Animal models for examining human breast cancer (HBC) carcinogenesis have been extensively studied and proposed. With the recent advent of immunotherapy, significant attention has been focused on the dog as a model for human cancer. Dogs develop mammary tumors and other cancer types spontaneously with an intact immune system, which exhibit a number of clinical and molecular similarities to HBC. In addition to the spontaneous tumor presentation, the clinical similarities between human and canine mammary tumors (CMT) include the age at onset, hormonal etiology and course of the diseases. Furthermore, factors that affect the disease outcome, including tumor size, stage and lymph node invasion, are similar in HBC and CMT. Similarly, the molecular characteristics of steroid receptor, epidermal growth factor, proliferation marker, metalloproteinase and cyclooxygenase expression, and the mutation of the p53 tumor suppressor gene in CMT, mimic HBC. Furthermore, ductal carcinomas in situ in human and canine mammary glands are particularly similar in their pathological, molecular and visual characteristics. These CMT characteristics and their similarities to HBC indicate that the dog could be an excellent model for the study of human disease. These similarities are discussed in detail in the present review, and are compared with the in vitro and other in vivo animal models available.

Genotoxicity profiles in exfoliated human mammary cells recovered from lactating mothers in Istanbul; relationship with demographic and dietary factors.

PubMed

Yilmaz, Bayram; Sandal, Suleyman; Ayvaci, Habibe; Tug, Niyazi; Vitrinel, Ayca

2012-12-12

We have investigated the presence of DNA damage in human mammary epithelial cells collected from healthy lactating mothers (age, 20-35 years) who were resident in the Istanbul area. Breast milk (10ml) was collected from 30 women between one and two weeks post-partum. Demographic information (parity, breast cancer, occupation, duration of residency in Istanbul, consumption of fish, beef and poultry) was also obtained. Milk samples were diluted 1:1 with RPMI 1640 medium and centrifuged to collect cells. The cells were re-suspended and cell viability was determined by use of 0.4% trypan blue. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). Fifty cells per slide and two slides per sample were scored to evaluate DNA damage. The cells were visually classified into four categories on the basis of extent of migration: undamaged (UD), lightly damaged (LD), moderately damaged (MD) and highly damaged (HD). Total comet scores (TCS) were calculated as: 1× UD+2× LD+3× MD+4× HD. Exfoliated mammary cells of the donors showed high (TCS≥150a.u.), moderate and low DNA damage in 10 (33.3%), 8 (26.7%) and 12 (40%) mothers, respectively. There was no significant correlation between TCS for DNA damage and the duration of previous breastfeeding, parity or age. None of the mothers was vegetarian, smoker or on any medication. Meat and chicken consumption did not significantly correlate with the TCS values. Fish consumption was significantly correlated with TCS results (Spearman's rho=0.39, p<0.05). No significant correlation was found between the DNA-damage scores and the period of residency in Istanbul, but fish consumption increased as the duration of stay was longer (Spearman's rho=0.53, p<0.01). These findings suggest that the primary causes of differences in genotoxicity detected in lactating mothers in Istanbul may be of dietary origin. Our experience also confirms that sampling breast milk from lactating mothers provides a valuable

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer

PubMed Central

Miwa, Hazuki E; Koba, Wade R; Fine, Eugene J; Giricz, Orsi; Kenny, Paraic A; Stanley, Pamela

2013-01-01

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3−/−/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3−/−/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer. PMID:24037315

Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress.

PubMed

Hein, S M; Haricharan, S; Johnston, A N; Toneff, M J; Reddy, J P; Dong, J; Bu, W; Li, Y

2016-03-17

In the normal mammary gland, the basal epithelium is known to be bipotent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bipotent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in-vivo lineage-tracing work demonstrates that luminal cells are capable of producing basal cells on activation of either polyoma middle T antigen or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer.

Luminal Epithelial Cells within the Mammary Gland Can Produce Basal Cells upon Oncogenic Stress

PubMed Central

Hein, Sarah M.; Haricharan, Svasti; Johnston, Alyssa N.; Toneff, Michael J.; Reddy, Jay P.; Dong, Jie; Bu, Wen; Li, Yi

2015-01-01

In the normal mammary gland, the basal epithelium is known to be bi-potent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bi-potent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here, we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in vivo lineage tracing work demonstrates that luminal cells are capable of producing basal cells upon activation of either Polyoma Middle T antigen (PyMT) or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer. PMID:26096929

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

PubMed Central

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 those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell

The Mammary Stem Cell Hierarchy: A Looking Glass into Heterogeneous Breast Cancer Landscapes

PubMed Central

Sreekumar, Amulya; Roarty, Kevin; Rosen, Jeffrey M.

2015-01-01

The mammary gland is a dynamic organ that undergoes extensive morphogenesis during the different stages of embryonic development, puberty, estrus, pregnancy, lactation and involution. Systemic and local cues underlie this constant tissue remodeling and act by eliciting an intricate pattern of responses in the mammary epithelial and stromal cells. Decades of studies utilizing methods such as transplantation and lineage tracing have identified a complex hierarchy of mammary stem cells, progenitors and differentiated epithelial cells that fuel mammary epithelial development. Importantly, these studies have extended our understanding of the molecular crosstalk between cell types, and signaling pathways maintaining normal homeostasis that often are deregulated during tumorigenesis. While several questions remain, this research has many implications for breast cancer. Fundamental among these are the identification of the cells of origin for the multiple subtypes of breast cancer and the understanding of tumor heterogeneity. A deeper understanding of these critical questions will unveil novel breast cancer drug targets and treatment paradigms. In this review, we provide a current overview of normal mammary development and tumorigenesis from a stem cell perspective. PMID:26206777

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

PubMed

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

2015-11-05

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. Copyright © 2015 Elsevier B.V. All rights reserved.

[Proliferative activity of cells in dyshormonal fibroadenomatosis of the human breast].

PubMed

Gudim-Levkovich, K A; Iakhimovich, L V; Slinchak, S M; Kaminskaia, L P; Kovbasiuk, S A

1981-11-01

Fibroadenomatous tissue of the human mammary gland was cultivated in diffuse chambers implanted into mice. On day 6 of culture the growing cells were subjected to morphological and autoradiographic analysis. The index of 3H-thymidine labeling of cell nuclei was found to correlate with the morphological pattern of dyshormonal fibroadenomatosis of the mammary gland. It is discussed whether it is desirable to use the culture in diffuse chambers for screening the actively proliferating forms human mammary gland dyshormonal dysplasias prone to malignancy.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Plasticity of mammary development in the prepubertal bovine mammary gland.

PubMed

Akers, R M

2017-12-01

Although peripubertal mammary development represents only a small fraction of the total mass of mammary parenchyma present in the udder at the end of gestation and into lactation, there is increasing evidence that the tissue foundations created in early life can affect future mammary development and function. Studies on expression of estrogen and progesterone receptors seem to confirm the relevance of these steroids in prepubertal mammary development, but connections with other growth factors, hormones, and local tissue factors remain elusive. Enhanced preweaning feeding in the bovine appears to enhance the capacity of mammary tissue to response to mammogenic stimulation. This suggests the possibility that improved early nutrition might allow for creation of stem or progenitor cell populations to better support the massive ductal growth and lobulo-alveolar development during gestation. Increasing evidence that immune cells are involved in mammary development suggests there are unexpected and poorly understood connections between the immune system and mammary development. This is nearly unexplored in ruminants. Development of new tools to identify, isolate, and characterize cell populations within the developing bovine mammary gland offer the possibility of identifying and perhaps altering populations of mammary stem cells or selected progenitor cells to modulate mammary development and, possibly, mammary function.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial.

PubMed

Pharo, Elizabeth A; Renfree, Marilyn B; Cane, Kylie N

2016-11-15

The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, Martha R.

1989-01-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, M.R.

1985-07-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

TRIM24 links glucose metabolism with transformation of human mammary epithelial cells.

PubMed

Pathiraja, T N; Thakkar, K N; Jiang, S; Stratton, S; Liu, Z; Gagea, M; Shi, X; Shah, P K; Phan, L; Lee, M-H; Andersen, J; Stampfer, M; Barton, M C

2015-05-28

Tripartite motif 24 protein (TRIM24) is a plant homeodomain/bromodomain histone reader, recently associated with poor overall survival of breast-cancer patients. At a molecular level, TRIM24 is a negative regulator of p53 levels and a co-activator of estrogen receptor. However, the role of TRIM24 in breast tumorigenesis remains largely unknown. We used an isogenic human mammary epithelial cell (HMEC) culture model, derived from reduction mammoplasty tissue, and found that ectopic expression of TRIM24 in immortalized HMECs (TRIM24 iHMECs) greatly increased cellular proliferation and induced malignant transformation. Subcutaneous injection of TRIM24 iHMECs in nude mice led to growth of intermediate to high-grade tumors in 60-70% of mice. Molecular analysis of TRIM24 iHMECs revealed a glycolytic and tricarboxylic acid cycle gene signature, alongside increased glucose uptake and activated aerobic glycolysis. Collectively, these results identify a role for TRIM24 in breast tumorigenesis through reprogramming of glucose metabolism in HMECs, further supporting TRIM24 as a viable therapeutic target in breast cancer.

CLOCK regulates mammary epithelial cell growth and differentiation

PubMed Central

Crodian, Jennifer; Suárez-Trujillo, Aridany; Erickson, Emily; Weldon, Bethany; Crow, Kristi; Cummings, Shelby; Chen, Yulu; Shamay, Avi; Mabjeesh, Sameer J.; Plaut, Karen

2016-01-01

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland. PMID:27707717

Mammary stem cells: Novel markers and novel approaches to increase lactation efficiency

USDA-ARS?s Scientific Manuscript database

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...

Evaluation of STAT5A Gene Expression in Aflatoxin B1 Treated Bovine Mammary Epithelial Cells

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

Purpose: Aflatoxin B1 (AFB1) is a potent mycotoxin which has been produced by fungi such as Aspergillus flavus and Aspergillus parasiticus as secondary metabolites due to their growth on food stuffs and induces hepatocellular carcinoma in many animal species, including humans. In the present study, the effect of AFB1 on STAT5A gene expression was investigated in bovine mammary epithelial cells using real time RT-PCR. Methods: Bovine mammary epithelial 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, cells were treated with AFB1 and incubated for 8 h. For real time PCR reaction, total RNA from the cultured and treated cells was extracted and used for complementary DNA synthesis. Results: The expression of STAT5A gene was significantly down regulated by AFB1 in dose- dependent manner and led to the reduction of proliferation and differentiation of epithelial cells, which has direct effect in milk protein quantity and quality. Conclusion: According to the results, it seems that down regulation of STAT5A gene in AFB1-treated cells maybe due to DNA damage induced by AFB1 in bovine mammary epithelial cells. PMID:24312879

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2.

PubMed

Beirão, Breno C B; Raposo, Teresa; Pang, Lisa Y; Argyle, David J

2015-07-15

Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma. We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes. Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators.

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

PubMed

Lin, Ye; Sun, Xiaoxu; Hou, Xiaoming; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-05-26

Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, β4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and β4GalT-I. Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.

Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis.

PubMed

Visvader, Jane E

2009-11-15

The epithelium of the mammary gland exists in a highly dynamic state, undergoing dramatic morphogenetic changes during puberty, pregnancy, lactation, and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed, with implications for understanding the cellular etiology underpinning breast tumor heterogeneity.

Prevention of Human Mammary Carcinogenesis

DTIC Science & Technology

1995-06-30

selected naturally-occurring agents (-)- epigallocatechin gallate ( EGCG ), indole-3-carbinol (13C) and genistein (GEN) for growth inhibition of 184-B5...mechanisms of BP-induced and GEN-induced alterations in cell cycle are being investigated in the ongoing studies. In addition, effects of 13C and EGCG are...rodent mammary tumorigenesis. The maximally nontoxic doses of EGCG , 13C and GEN identified by initial dose-response experiments, were used. The data

Lgr4 regulates mammary gland development and stem cell activity through the pluripotency transcription factor Sox2.

PubMed

Wang, Ying; Dong, Jie; Li, Dali; Lai, Li; Siwko, Stefan; Li, Yi; Liu, Mingyao

2013-09-01

The key signaling networks regulating mammary stem cells are poorly defined. The leucine-rich repeat containing G protein-coupled receptor (Lgr) family has been implicated in intestinal, gastric, and epidermal stem cell functions. We investigated whether Lgr4 functions in mammary gland development and mammary stem cells. We found that Lgr4(-/-) mice had delayed ductal development, fewer terminal end buds, and decreased side-branching. Crucially, the mammary stem cell repopulation capacity was severely impaired. Mammospheres from Lgr4(-/-) mice showed decreased Wnt signaling. Wnt3a treatment prevented the adverse effects of Lgr4 loss on organoid formation. Chromatin immunoprecipitation analysis indicated that Sox2 expression was controlled by the Lgr4/Wnt/β-catenin/Lef1 pathway. Importantly, Sox2 overexpression restored the in vivo mammary regeneration potential of Lgr4(-/-) mammary stem cells. Therefore, Lgr4 activates Sox2 to regulate mammary development and stem cell functions via Wnt/β-catenin/Lef1. © AlphaMed Press.

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

PubMed

Horibata, Sachi; Rogers, Katherine E; Sadegh, David; Anguish, Lynne J; McElwee, John L; Shah, Pragya; Thompson, Paul R; Coonrod, Scott A

2017-05-26

Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

PubMed

Barbieri, Federica; Thellung, Stefano; Ratto, Alessandra; Carra, Elisa; Marini, Valeria; Fucile, Carmen; Bajetto, Adriana; Pattarozzi, Alessandra; Würth, Roberto; Gatti, Monica; Campanella, Chiara; Vito, Guendalina; Mattioli, Francesca; Pagano, Aldo; Daga, Antonio; Ferrari, Angelo; Florio, Tullio

2015-04-07

Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Similarly to the human counterpart, CMCs contain stem

Regulation of gene expression in human mammary epithelium: effect of breast pumping

USDA-ARS?s Scientific Manuscript database

Little is known of the molecular regulation of human milk production because of limitations in obtaining mammary tissue from lactating women. Our objectives were to evaluate whether RNA isolated from breast milk fat globules (MFGs) could be an alternative to mammary biopsies and to determine whether...

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

PubMed

Tong, Hui-Li; Li, Qing-Zhang; Gao, Xue-Jun; Yin, De-Yun

2012-03-01

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Mammary Stem Cells and Breast Cancer Stem Cells: Molecular Connections and Clinical Implications.

PubMed

Celià-Terrassa, Toni

2018-05-04

Cancer arises from subpopulations of transformed cells with high tumor initiation and repopulation ability, known as cancer stem cells (CSCs), which share many similarities with their normal counterparts. In the mammary gland, several studies have shown common molecular regulators between adult mammary stem cells (MaSCs) and breast cancer stem cells (bCSCs). Cell plasticity and self-renewal are essential abilities for MaSCs to maintain tissue homeostasis and regenerate the gland after pregnancy. Intriguingly, these properties are similarly executed in breast cancer stem cells to drive tumor initiation, tumor heterogeneity and recurrence after chemotherapy. In addition, both stem cell phenotypes are strongly influenced by external signals from the microenvironment, immune cells and supportive specific niches. This review focuses on the intrinsic and extrinsic connections of MaSC and bCSCs with clinical implications for breast cancer progression and their possible therapeutic applications.

Mouse Mammary Tumor Virus c-rel Transgenic Mice Develop Mammary Tumors

PubMed Central

Romieu-Mourez, Raphaëlle; Kim, Dong W.; Min Shin, Sang; Demicco, Elizabeth G.; Landesman-Bollag, Esther; Seldin, David C.; Cardiff, Robert D.; Sonenshein, Gail E.

2003-01-01

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-κB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-κB. Analysis of the composition of NF-κB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-κB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-κB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary

Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

PubMed Central

Gentile, Luciana B.; Nagamine, Marcia K.; Biondi, Luiz R.; Sanches, Daniel S.; Toyota, Fábio; Giovani, Tatiane M.; de Jesus, Isis P.; da Fonseca, Ivone I. M.; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L.; Salles Gomes, Cristina de O. Massoco

2017-01-01

There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development. PMID:28945747

Precursors of hexoneogenesis within the human mammary gland

USDA-ARS?s Scientific Manuscript database

The human mammary gland is capable of de novo synthesis of glucose and galactose (hexoneogenesis); however, the carbon source is incompletely understood. In this study, we investigated the role of acetate, glutamine, lactate and glycerol as potential carbon sources for hexoneogenesis. Healthy breast...

Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia.

PubMed

Li, Xin; Gonzalez, Maria E; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D; Kleer, Celina G

2009-09-01

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

Targeted Overexpression of EZH2 in the Mammary Gland Disrupts Ductal Morphogenesis and Causes Epithelial Hyperplasia

PubMed Central

Li, Xin; Gonzalez, Maria E.; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D.; Kleer, Celina G.

2009-01-01

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with β-catenin, inducing β-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/β-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with β-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma. PMID:19661437

CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

PubMed

Wiehle, Ronald; Lantvit, Daniel; Yamada, Tohru; Christov, Konstantin

2011-03-01

CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P < 0.02), decreased incidence from 85% to 35% (P < 0.001), and reduced multiplicity from 3.0 to 1.1 tumors/animal (P < 0.001). Tumor burden decreased from 2.6 g/animal to 0.26 g/animal (P < 0.01). CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il; The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem; Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il

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.more » 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.« less

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

PubMed

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. Copyright © 2012 Elsevier Inc. All rights reserved.

Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

PubMed

Wang, Y; Baumrucker, C R

2010-07-01

Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (P<0.05) and exhibited a dose-dependent inhibition with 9-cisRA (9cRA; P<0.05). The MeBo cells exhibited some inhibition with these natural ligands (P<0.05), but they were not as sensitive. The addition of bLf had similar inhibitory effects (P<0.05) on cell viability of the 2 mammary cell types. Applications of RA receptor (RAR) agonist indicated that the stimulation of the RAR in both mammary cell types was highly effective in inhibition of cell viability (P<0.05), whereas the application of an RAR antagonist stimulated MeBo cell viability (P<0.05) and inhibited BME-UV1 cell viability (P<0.05). Finally, the use of the RAR antagonist in conjunction with bLf indicated a rescue of the bLf effect in the MeBo cells, suggesting that bLf is acting through the RAR receptor. Conversely, bLf reverted inhibition of cell viability by 9cRA in the BME-UV1 cell type (P<0.05). We conclude that RAR interaction in bovine mammary cell types regulates cell viability in vitro; we hypothesize that the natural ligands mediate regulation of bovine mammary cell viability in vivo and that bLf can either enhance or reverse the retinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

PubMed Central

Fang, Mingzhu; Guo, Wei-Ren; Park, Youngil; Kang, Hwan-Goo; Zarbl, Helmut

2015-01-01

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis. PMID:26544624

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

NASA Astrophysics Data System (ADS)

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-04-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53-/- mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53-/- mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

PubMed Central

Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

2014-01-01

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53−/− mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53−/− mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers. PMID:24722541

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli.

PubMed

Munne, Pauliina M; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G

2014-04-11

Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

Development of human epithelial cell systems for radiation risk assessment

NASA Astrophysics Data System (ADS)

Yang, C. H.; Craise, L. M.

1994-10-01

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

PubMed Central

Godlewska, Marlena; Krasuska, Wanda

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes. PMID:29513734

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

PubMed

Godlewska, Marlena; Krasuska, Wanda; Czarnocka, Barbara

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Differential Gene Expression in Normal Human Mammary Epithelial Cells Treated with Malathion Monitored by DNA Microarrays

PubMed Central

Gwinn, Maureen R.; Whipkey, Diana L.; Tennant, Lora B.; Weston, Ainsley

2005-01-01

Organophosphate pesticides are a major source of occupational exposure in the United States. Moreover, malathion has been sprayed over major urban populations in an effort to control mosquitoes carrying West Nile virus. Previous research, reviewed by the U.S. Environmental Protection Agency, on the genotoxicity and carcinogenicity of malathion has been inconclusive, although malathion is a known endocrine disruptor. Here, interindividual variations and commonality of gene expression signatures have been studied in normal human mammary epithelial cells from four women undergoing reduction mammoplasty. The cell strains were obtained from the discarded tissues through the Cooperative Human Tissue Network (sponsors: National Cancer Institute and National Disease Research Interchange). Interindividual variation of gene expression patterns in response to malathion was observed in various clustering patterns for the four cell strains. Further clustering identified three genes with increased expression after treatment in all four cell strains. These genes were two aldo–keto reductases (AKR1C1 and AKR1C2) and an estrogen-responsive gene (EBBP). Decreased expression of six RNA species was seen at various time points in all cell strains analyzed: plasminogen activator (PLAT), centromere protein F (CPF), replication factor C (RFC3), thymidylate synthetase (TYMS), a putative mitotic checkpoint kinase (BUB1), and a gene of unknown function (GenBank accession no. AI859865). Expression changes in all these genes, detected by DNA microarrays, have been verified by real-time polymerase chain reaction. Differential changes in expression of these genes may yield biomarkers that provide insight into interindividual variation in malathion toxicity. PMID:16079077

GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells.

PubMed

Xu, Gaoxiao; Duan, Saixing; Hou, Jianye; Wei, Zhongxin; Zhao, Guangwei

2017-01-01

It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase (FASN) , stearoyl-CoA desaturase (SCD) , fatty acid binding protein 4 (FABP4) , diacylglycerol acyltransferase 1 (DGAT1) , perilipin 2 (PLIN2) , perilipin 3 (PLIN3) , and C/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPAR γ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression of Bcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPAR γ in mouse mammary glands.

Expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk.

PubMed

Murakami, Masamoto; Dorschner, Robert A; Stern, Lauren J; Lin, Kenneth H; Gallo, Richard L

2005-01-01

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.

The RhoGEF Net1 Is Required for Normal Mammary Gland Development

PubMed Central

Zuo, Yan; Berdeaux, Rebecca

2014-01-01

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. PMID:25321414

(Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meeuwen, J.A. van; Korthagen, N.; Jong, P.C. de

In the public opinion, phytochemicals (PCs) present in the human diet are often considered beneficial (e.g. by preventing breast cancer). Two possible mechanisms that could modulate tumor growth are via interaction with the estrogen receptor (ER) and inhibition of aromatase (CYP19). Multiple in vitro studies confirmed that these compounds act estrogenic, thus potentially induce tumor growth, as well as aromatase inhibitory, thus potentially reduce tumor growth. It is thought that in the in vivo situation breast epithelial (tumor) cells communicate with surrounding connective tissue by means of cytokines, prostaglandins and estradiol forming a complex feedback mechanism. Recently our laboratory developedmore » an in vitro co-culture model of healthy mammary fibroblasts and MCF-7 cells that (at least partly) simulated this feedback mechanism (M. Heneweer et al., TAAP vol. 202(1): 50-58, 2005). In the present study biochanin A, chrysin, naringenin, apigenin, genistein and quercetin were studied for their estrogenic properties (cell proliferation, pS2 mRNA) and aromatase inhibition in MCF-7 breast tumor cells, healthy mammary fibroblasts and their co-culture. The proliferative potency of these compounds in the MCF-7 cells derived from their EC{sub 50}s decreased in the following order: estadiol (4*10{sup -3} nM) > biochanin A (9 nM) > genistein (32 nM) > testosterone (46 nM) > naringenin (287 nM) > apigenin (440 nM) > chrysin (4 {mu}M). The potency to inhibit aromatase derived from their IC{sub 50}s decreased in the following order: chrysin (1.5 {mu}M) > naringenin (2.2 {mu}M) > genistein (3.6 {mu}M) > apigenin (4.1 {mu}M) > biochanin A (25 {mu}M) > quercetin (30 {mu}M). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1-10 {mu}M). Results from co-cultures did not elucidate the dominant effect of these compounds. MCF-7 cell proliferation occurs at concentrations that are not uncommon

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Rohit B.; Wang, Qingde; Khillan, Jaspal S., E-mail: khillan@pitt.edu

Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibitmore » mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.« less

Expression of novel, putative stem cell markers in prepubertal and lactating mammary glands of bovine

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) are essential for growth and maintenance of the mammary epithelium. Two main phases of mammary growth include ductal elongation prior to puberty and lobulo-alveolar growth and development during pregnancy. Some studies have utilized morphological characteristics and retenti...

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

PubMed

Sokol, Ethan S; Miller, Daniel H; Breggia, Anne; Spencer, Kevin C; Arendt, Lisa M; Gupta, Piyush B

2016-03-01

Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell

Development of human epithelial cell systems for radiation risk assessment

NASA Technical Reports Server (NTRS)

Yang, C. H.; Craise, L. M.

1994-01-01

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells

PubMed Central

Benavente, Micaela Andrea; Bianchi, Carolina Paula; Imperiale, Fernanda; Aba, Marcelo Alfredo

2016-01-01

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient’s overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 103 and 8 × 103 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 103 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 103 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 103 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 103 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis. PMID:28083539

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells.

PubMed

Benavente, Micaela Andrea; Bianchi, Carolina Paula; Imperiale, Fernanda; Aba, Marcelo Alfredo

2016-01-01

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient's overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 10 3 and 8 × 10 3 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 10 3 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 10 3 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 10 3 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 10 3 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis.

Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture.

PubMed

Zeng, Yi Arial; Nusse, Roel

2010-06-04

Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. In addition, stem cells tend to differentiate in the absence of the proper signals and are therefore difficult to maintain in cell culture. The mammary gland provides an excellent system to study self-renewal signals, because the organ develops postnatally, arises from stem cells, and is readily generated from transplanted cells. We show here that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In addition, stem cells mutant for the negative-feedback regulator Axin2 and therefore sensitized to Wnt signals have a competitive advantage in mammary gland reconstitution assays. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We conclude that Wnt proteins serve as rate-limiting self-renewal signals acting directly on mammary stem cells. Copyright 2010 Elsevier Inc. All rights reserved.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

ApcMin, A Mutation in the Murine Apc Gene, Predisposes to Mammary Carcinomas and Focal Alveolar Hyperplasias

NASA Astrophysics Data System (ADS)

Moser, Amy Rapaich; Mattes, Ellen M.; Dove, William F.; Lindstrom, Mary J.; Haag, Jill D.; Gould, Michael N.

1993-10-01

ApcMin (Min, multiple intestinal neoplasia) is a point mutation in the murine homolog of the APC gene. Min/+ mice develop multiple intestinal adenomas, as do humans carrying germ-line mutations in APC. Female mice carrying Min are also prone to develop mammary tumors. Min/+ mammary glands are more sensitive to chemical carcinogenesis than are +/+ mammary glands. Transplantation of mammary cells from Min/+ or +/+ donors into +/+ hosts demonstrates that the propensity to develop mammary tumors is intrinsic to the Min/+ mammary cells. Long-term grafts of Min/+ mammary glands also gave rise to focal alveolar hyperplasias, indicating that the presence of the Min mutation also has a role in the development of these lesions.

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

PubMed

He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

2017-01-01

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montesano, Roberto; Sarkoezi, Rita; Schramek, Herbert

2008-09-12

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hithertomore » unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.« less

PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.

PubMed

Lin, Meng-Chieh; Chen, Shih-Yin; Tsai, Ho-Min; He, Pei-Lin; Lin, Yen-Chun; Herschman, Harvey; Li, Hua-Jung

2017-02-01

Prostaglandin E 2 (PGE 2 )-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE 2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE 2 /prostaglandin E receptor 4 (EP 4 ) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP 4 ) antagonism. EP 4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP 4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP 4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP 4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP 4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE 2 /EP 4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP 4 -mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. Stem Cells 2017;35:425-444. © 2016 The Authors STEM CELLS

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

PubMed

Cyr, A R; Kulak, M V; Park, J M; Bogachek, M V; Spanheimer, P M; Woodfield, G W; White-Baer, L S; O'Malley, Y Q; Sugg, S L; Olivier, A K; Zhang, W; Domann, F E; Weigel, R J

2015-01-22

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity.

PubMed

Wanyonyi, Stephen S; Sharp, Julie A; Khalil, Elie; Lefevre, Christophe; Nicholas, Kevin R

2011-11-01

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

Unravelling the mystery of stem/progenitor cells in human breast milk.

PubMed

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A; Cregan, Mark D; Chan, Jerry K Y

2010-12-28

Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6 ± 0.8% (mean ± SEM) and 0.7 ± 0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8 ± 9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17 ± 0.2% and 0.9 ± 0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8 ± 0.4% of WCP) as well as CD133+ cells (1.7 ± 0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6 ± 8.6 vs 18.1 ± 6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman.

Unravelling the Mystery of Stem/Progenitor Cells in Human Breast Milk

PubMed Central

Fan, Yiping; Chong, Yap Seng; Choolani, Mahesh A.; Cregan, Mark D.; Chan, Jerry K. Y.

2010-01-01

Background Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. Methodology/Principal Findings We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6±0.8% (mean±SEM) and 0.7±0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8±9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17±0.2% and 0.9±0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8±0.4% of WCP) as well as CD133+ cells (1.7±0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6±8.6 vs 18.1±6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. Conclusions/Significance The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman. PMID:21203434

Fingerprinting Breast Cancer vs. Normal Mammary Cells by Mass Spectrometric Analysis of Volatiles

NASA Astrophysics Data System (ADS)

He, Jingjing; Sinues, Pablo Martinez-Lozano; Hollmén, Maija; Li, Xue; Detmar, Michael; Zenobi, Renato

2014-06-01

There is increasing interest in the development of noninvasive diagnostic methods for early cancer detection, to improve the survival rate and quality of life of cancer patients. Identification of volatile metabolic compounds may provide an approach for noninvasive early diagnosis of malignant diseases. Here we analyzed the volatile metabolic signature of human breast cancer cell lines versus normal human mammary cells. Volatile compounds in the headspace of conditioned culture medium were directly fingerprinted by secondary electrospray ionization-mass spectrometry. The mass spectra were subsequently treated statistically to identify discriminating features between normal vs. cancerous cell types. We were able to classify different samples by using feature selection followed by principal component analysis (PCA). Additionally, high-resolution mass spectrometry allowed us to propose their chemical structures for some of the most discriminating molecules. We conclude that cancerous cells can release a characteristic odor whose constituents may be used as disease markers.

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Stromal adipocyte enhancer-binding protein (AEBP1) promotes mammary epithelial cell hyperplasia via proinflammatory and hedgehog signaling.

PubMed

Holloway, Ryan W; Bogachev, Oleg; Bharadwaj, Alamelu G; McCluskey, Greg D; Majdalawieh, Amin F; Zhang, Lei; Ro, Hyo-Sung

2012-11-09

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

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

PubMed Central

Holloway, Ryan W.; Bogachev, Oleg; Bharadwaj, Alamelu G.; McCluskey, Greg D.; Majdalawieh, Amin F.; Zhang, Lei; Ro, Hyo-Sung

2012-01-01

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1TG bone marrow cells into non-transgenic (AEBP1NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1TG mammary macrophages and epithelium. Shh expression was induced in AEBP1TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1TG peritoneal macrophages. The conditioned AEBP1TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis. PMID:22995915

Cytotoxic effect of achatinin(H) (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7).

PubMed

Dharmu, Indra; Ramamurty, N; Kannan, Ramalingam; Babu, Mary

2007-01-01

The hemolymph-derived achatinin(H) (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC(50) values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin(H) ranged from 6 to 10 microg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 microg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin(H) treatment. The specificity and purity of the achatinin(H) was confirmed by the Western blot assay. Achatinin(H) binding to MCF7 cells was detected by anti-achatinin(H), and visualization of the achatinin(H) binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin(H) binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin(H) treatment. The cells were arrested in G(2)/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation

DTIC Science & Technology

2006-03-01

1-0322 TITLE: Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary Epithelial Cell Differentiation PRINCIPAL...Summary 3. DATES COVERED (From - To) 1 MAR 2005 - 28 FEB 2006 4. TITLE AND SUBTITLE Roles of Breast Cancer Susceptibility Genes BRCA’s in Mammary...reverse the phenotype of differentiation-defective breast cancer cells bearing reduced BRCA1 functions. This result implies BRCA1 is involved in

Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

PubMed

Nagahara, Rei; Kimura, Masayuki; Itahashi, Megu; Sugahara, Go; Kawashima, Masashi; Murayama, Hirotada; Yoshida, Toshinori; Shibutani, Makoto

2016-11-01

Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas. © 2016 The Author(s).

Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

PubMed

Sánchez-Céspedes, R; Maniscalco, L; Iussich, S; Martignani, E; Guil-Luna, S; De Maria, R; Martín de Las Mulas, J; Millán, Y

2013-08-01

Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Ets transcription factor Elf5 specifies mammary alveolar cell fate

PubMed Central

Oakes, Samantha R.; Naylor, Matthew J.; Asselin-Labat, Marie-Liesse; Blazek, Katrina D.; Gardiner-Garden, Margaret; Hilton, Heidi N.; Kazlauskas, Michael; Pritchard, Melanie A.; Chodosh, Lewis A.; Pfeffer, Peter L.; Lindeman, Geoffrey J.; Visvader, Jane E.; Ormandy, Christopher J.

2008-01-01

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development. PMID:18316476

Genotoxic Exposure during Juvenile Growth of Mammary Gland Depletes Stem Cell Activity and Inhibits Wnt Signaling

PubMed Central

Klos, Kristine S.; Kim, Soyoung; Alexander, Caroline M.

2012-01-01

Various types of somatic stem cell have been tested for their response to genotoxic exposure, since these cells are likely to be important to regeneration, aging and cancer. In this study, we evaluated the response of mammary stem cells to genotoxic exposure during ductal growth in juveniles. Exposure to the polycyclic aromatic hydrocarbon (DMBA; 7,12 dimethylbenz[a]anthracene) had no gross effect on outgrowth and morphogenesis of the ductal tree, or upon lobuloalveolar growth during pregnancy. However, by fat pad assay, we found that mammary stem cell activity was reduced by 80% in glands from adults that were exposed to genotoxins as juveniles. The associated basal cell lineage was depleted. Both basal and luminal cells showed a robust response to genotoxic exposure (including γH2AX phosphorylation, pS15p53 and pT68Chk2), with durable hyperproliferation, but little cytotoxicity. Since the phenotype of these glands (low basal cell fraction, low stem cell activity) phenocopies mammary glands with loss of function for Wnt signaling, we measured Wnt signaling in genotoxin-exposed glands, and found a durable reduction in the activation of the canonical signaling Wnt receptors, Lrp5/6. Furthermore, when mammary epithelial cells were treated with Wnt3a, DMBA exposure reduced the basal cell population and Lrp activation was ablated. We conclude that during active ductal growth, Wnt-dependent mammary stem cells are sensitized to cell death by genotoxin exposure. Our conclusion may be important for other tissues, since all solid tumor stem cell activities have been shown to be Wnt-dependent to date. PMID:23185480

The origin of human milk bacteria: is there a bacterial entero-mammary pathway during late pregnancy and lactation?

PubMed

Rodríguez, Juan M

2014-11-01

Human milk is a source of bacteria to the infant gut; however, the origin of milk bacteria, as well as their impact on neonatal gut microbiota establishment, remains largely unknown. In the past years, results provided by different research groups suggest that certain bacteria from the maternal gastrointestinal tract could translocate through a mechanism involving mononuclear immune cells, migrate to the mammary glands via an endogenous cellular route (the bacterial entero-mammary pathway), and subsequently colonize the gastrointestinal tract of the breast-fed neonate. If such findings are confirmed in the future, we could exert a positive influence on infant health by modulating the maternal gut microbiota. © 2014 American Society for Nutrition.

Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

DTIC Science & Technology

2015-12-01

Our major goals are to determine whether Fetal Mammary Stem Cell (fMaSC) signatures correlate with response to chemotherapy and metastasis in...these aims will enable us to: 1) better categorize distinct cell types within the fMaSC population, 2) identify biomarkers for prospective stem cell purification...and in situ localization, and 3) identify candidate stem cell regulatory pathways that should reveal therapeutic targets and improved

Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

DTIC Science & Technology

2014-10-01

Our major goals are to determine whether Fetal Mammary Stem Cell (fMaSC) signatures correlate with response to chemotherapy and metastasis in...these aims will enable us to: 1) better categorize distinct cell types within the fMaSC population, 2) identify biomarkers for prospective stem cell purification...and in situ localization, and 3) identify candidate stem cell regulatory pathways that should reveal therapeutic targets and improved

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

PubMed

Sánchez, O; Montesino, R; Toledo, J R; Rodríguez, E; Díaz, D; Royle, L; Rudd, P M; Dwek, R A; Gerwig, G J; Kamerling, J P; Harvey, D J; Cremata, J A

2007-08-15

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

PubMed

Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

2018-06-01

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

PubMed

Ishikawa, F; Ushida, K; Mori, K; Shibanuma, M

2015-01-22

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

PubMed

Avdulov, Svetlana; Li, Shunan; Michalek, Van; Burrichter, David; Peterson, Mark; Perlman, David M; Manivel, J Carlos; Sonenberg, Nahum; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2004-06-01

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

A simple ductal mammary papilloma in a male maned wolf (Chrysocyon brachyurus).

PubMed

Cassali, Geovanni D; Bertagnolli, Angélica C; Ferreira, Enio; Malta, Marcelo C C

2009-01-01

A 1-cm-diameter nodule was identified in the left inguinal mammary gland of a 9-year-old male maned wolf (Chrysocyon brachyurus). The mass was surgically excised and examined histologically. Microscopically, the neoplasm consisted of papillary proliferations of epithelial cells on well-defined fibrovascular stalks. A myoepithelial layer was located between the single layer of epithelial cells and the fibrovascular stalk. This histologic appearance was compatible with a diagnosis of simple ductal mammary papilloma. Immunohistochemical staining was positive for p63, cytokeratins AE1/AE3, and estrogen receptors. The clinical and histologic observations in the present case indicate that male maned wolves may develop mammary tumors that are similar to those observed in domestic dogs and humans.

Canonical Wnt Signaling as a Specific Marker for Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2012-02-01

These cells were identified by  flow   cytometry  to detect cells that were positive for CD24 and CD49f.   2. We have established that activation of Wnt...09-1-0072 TITLE: Canonical Wnt Signaling as a Specific marker for Normal and Tumorigenic Mammary Stem Cells PRINCIPAL...activation of canonical Wnt signaling may be a very specific marker for mammary stem cells and be a target for transformation that results in the

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Mammary Gland Development

PubMed Central

Macias, Hector

2012-01-01

The mammary gland develops through several distinct stages. The first transpires in the embryo as the ectoderm forms a mammary line that resolves into placodes. Regulated by epithelial/mesenchymal interactions, the placodes descend into the underlying mesenchyme and produce the rudimentary ductal structure of the gland present at birth. Subsequent stages of development – pubertal growth, pregnancy, lactation and involution – occur postnatally under the regulation of hormones. Puberty initiates branching morphogenesis, which requires growth hormone and estrogen, as well as IGF1, to create a ductal tree that fills the fat pad. Upon pregnancy the combined actions of progesterone and prolactin generate alveoli, which secrete milk during lactation. Lack of demand for milk at weaning initiates the process of involution whereby the gland is remodeled back to its pre-pregnancy state. These processes require numerous signaling pathways that have distinct regulatory functions at different stages of gland development. Signaling pathways also regulate a specialized subpopulation of mammary stem cells that fuel the dramatic changes in the gland occurring with each pregnancy. Our knowledge of mammary gland development and mammary stem cell biology has significantly contributed to our understanding of breast cancer and has advanced the discovery of therapies to treat this disease. PMID:22844349

Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

PubMed Central

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

2016-01-01

Human milk (HM) contains regulatory biomolecules including miRNAs, the origin and functional significance of which are still undetermined. We used TaqMan OpenArrays to profile 681 mature miRNAs in HM cells and fat, and compared them with maternal peripheral blood mononuclear cells (PBMCs) and plasma, and bovine and soy infant formulae. HM cells and PBMCs (292 and 345 miRNAs, respectively) had higher miRNA content than HM fat and plasma (242 and 219 miRNAs, respectively) (p < 0.05). A strong association in miRNA profiles was found between HM cells and fat, whilst PBMCs and plasma were distinctly different to HM, displaying marked inter-individual variation. Considering the dominance of epithelial cells in mature milk of healthy women, these results suggest that HM miRNAs primarily originate from the mammary epithelium, whilst the maternal circulation may have a smaller contribution. Our findings demonstrate that unlike infant formulae, which contained very few human miRNA, HM is a rich source of lactation-specific miRNA, which could be used as biomarkers of the performance and health status of the lactating mammary gland. Given the recently identified stability, uptake and functionality of food- and milk-derived miRNA in vivo, HM miRNA are likely to contribute to infant protection and development. PMID:26854194

Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk.

PubMed

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E; Geddes, Donna T; Kakulas, Foteini

2016-02-08

Human milk (HM) contains regulatory biomolecules including miRNAs, the origin and functional significance of which are still undetermined. We used TaqMan OpenArrays to profile 681 mature miRNAs in HM cells and fat, and compared them with maternal peripheral blood mononuclear cells (PBMCs) and plasma, and bovine and soy infant formulae. HM cells and PBMCs (292 and 345 miRNAs, respectively) had higher miRNA content than HM fat and plasma (242 and 219 miRNAs, respectively) (p < 0.05). A strong association in miRNA profiles was found between HM cells and fat, whilst PBMCs and plasma were distinctly different to HM, displaying marked inter-individual variation. Considering the dominance of epithelial cells in mature milk of healthy women, these results suggest that HM miRNAs primarily originate from the mammary epithelium, whilst the maternal circulation may have a smaller contribution. Our findings demonstrate that unlike infant formulae, which contained very few human miRNA, HM is a rich source of lactation-specific miRNA, which could be used as biomarkers of the performance and health status of the lactating mammary gland. Given the recently identified stability, uptake and functionality of food- and milk-derived miRNA in vivo, HM miRNA are likely to contribute to infant protection and development.

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

PubMed

Rauner, Gat; Barash, Itamar

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Mouse mammary tumor virus-like RNA transcripts and DNA are found in affected cells of human breast cancer.

PubMed

Ford, Caroline E; Faedo, Margaret; Rawlinson, William D

2004-11-01

Identifiable risk factors for the development of breast cancer include age, diet, family history, and lifetime estrogen exposure. An infectious agent (mouse mammary tumor virus; MMTV) is known to cause murine breast tumors and may be involved in the pathogenesis of human disease. Multiple studies have detected MMTV-like sequences in 30 to 60% of breast cancer samples and up to 1.8% of samples from normal breast. Using in situ PCR of MMTV-like sequences of formalin-fixed, paraffin-embedded breast tissue, viral sequences have been located in cancerous epithelial cells in breast acini of male and female breast tumors, but not in adjacent nonmalignant cells. MMTV-like sequences were also located in the epithelial cells of male gynecomastia samples. Using reverse transcriptase in situ PCR, RNA transcripts from the env gene were also detected within cancerous epithelial cells of 78% of DNA-positive tumors, 80% of gynecomastia samples, and 0% of normal tissues screened. This suggests the virus may be replicating in these cells. The epidemiologic and histopathological data are consistent with the association of an MMTV-like virus with breast cancers in men and women. The association with gynecomastia, a benign, possibly premalignant condition suggests hormonal influences, rather than cancer per se, may be the dominant factor in determining viral presence and replication.

Neem leaf extract inhibits mammary carcinogenesis by altering cell proliferation, apoptosis, and angiogenesis

PubMed Central

Arumugam, Arunkumar; Agullo, Pamela; Boopalan, Thiyagarajan; Nandy, Sushmita; Lopez, Rebecca; Gutierrez, Christina; Narayan, Mahesh; Rajkumar, Lakshmanaswamy

2014-01-01

Plant-based medicines are useful in the treatment of cancer. Many breast cancer patients use complementary and alternative medicine in parallel with conventional treatments. Neem is historically well known in Asia and Africa as a versatile medicinal plant with a wide spectrum of biological activities. The experiments reported herein determined whether the administration of an ethanolic fraction of Neem leaf (EFNL) inhibits progression of chemical carcinogen-induced mammary tumorigenesis in rat models. Seven-week-old female Sprague Dawley rats were given a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Upon the appearance of palpable mammary tumors, the rats were divided into vehicle-treated control groups and EFNL-treated groups. Treatment with EFNL inhibited MNU-induced mammary tumor progression. EFNL treatment was also highly effective in reducing mammary tumor burden and in suppressing mammary tumor progression even after the cessation of treatment. Further, we found that EFNL treatment effectively upregulated proapoptotic genes and proteins such as p53, B cell lymphoma-2 protein (Bcl-2)-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad) caspases, phosphatase and tensin homolog gene (PTEN), and c-Jun N-terminal kinase (JNK). In contrast, EFNL treatment caused downregulation of anti-apoptotic (Bcl-2), angiogenic proteins (angiopoietin and vascular endothelial growth factor A [VEGF-A]), cell cycle regulatory proteins (cyclin D1, cyclin-dependent kinase 2 [Cdk2], and Cdk4), and pro-survival signals such as NFκB, mitogen-activated protein kinase 1 (MAPK1). The data obtained in this study demonstrate that EFNL exert a potent anticancer effect against mammary tumorigenesis by altering key signaling pathways. PMID:24146019

The epithelial-mesenchymal transition generates cells with properties of stem cells.

PubMed

Mani, Sendurai A; Guo, Wenjun; Liao, Mai-Jing; Eaton, Elinor Ng; Ayyanan, Ayyakkannu; Zhou, Alicia Y; Brooks, Mary; Reinhard, Ferenc; Zhang, Cheng Cheng; Shipitsin, Michail; Campbell, Lauren L; Polyak, Kornelia; Brisken, Cathrin; Yang, Jing; Weinberg, Robert A

2008-05-16

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Pim-1 kinase expression during murine mammary development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gapter, Leslie A.; School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234; Magnuson, Nancy S.

2006-07-07

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile ofmore » progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21{sup CIP/Waf1}, and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.« less

CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy.

PubMed

Rostoker, Ran; Ben-Shmuel, Sarit; Rashed, Rola; Shen Orr, Zila; LeRoith, Derek

2016-05-14

The pro-tumorigenic effects of the insulin-like growth factor receptor (IGF1R) are well described. IGF1R promotes cancer cell survival and proliferation and prevents apoptosis, and, additionally it was shown that IGF1R levels are significantly elevated in most common human malignancies including breast cancer. However, results from phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are greatly warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies. Using the delivery of shRNA vectors into the Mvt1 cell line, we tested the role of the IGF1R in the development of mammary tumors. Based on CD24 cell surface expression, control and IGF1R-knockdown (IGF1R-KD) cells were FACS sorted into CD24(-) and CD24(+) subsets and further characterized in vitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF1R effect on the plasticity of this cell's phenotype. Metastatic capacity of the cells was assessed using the tail vein assay. In this study we demonstrate that downregulation of the IGF1R specifically in cancer cells expressing CD24 on the cell surface membrane affect both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF1R-KD abolished both CD24(+) cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG and a significant reduction of SLP1 expression in the CD24(+)/IGF1R-KD; tumor-suppressor and tumor-promoting genes respectively. Moreover, we demonstrate here that the IGF1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF1R-KD induces in vivo differentiation of the CD24(+) cells toward the

Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice.

PubMed

Lakritz, Jessica R; Poutahidis, Theofilos; Levkovich, Tatiana; Varian, Bernard J; Ibrahim, Yassin M; Chatzigiagkos, Antonis; Mirabal, Sheyla; Alm, Eric J; Erdman, Susan E

2014-08-01

Recent studies suggest health benefits including protection from cancer after eating fermented foods such as probiotic yogurt, though the mechanisms are not well understood. Here we tested mechanistic hypotheses using two different animal models: the first model studied development of mammary cancer when eating a Westernized diet, and the second studied animals with a genetic predilection to breast cancer. For the first model, outbred Swiss mice were fed a Westernized chow putting them at increased risk for development of mammary tumors. In this Westernized diet model, mammary carcinogenesis was inhibited by routine exposure to Lactobacillus reuteri ATCC-PTA-6475 in drinking water. The second model was FVB strain erbB2 (HER2) mutant mice, genetically susceptible to mammary tumors mimicking breast cancers in humans, being fed a regular (non-Westernized) chow diet. We found that oral supplement with these purified lactic acid bacteria alone was sufficient to inhibit features of mammary neoplasia in both models. The protective mechanism was determined to be microbially-triggered CD4+CD25+ lymphocytes. When isolated and transplanted into other subjects, these L. reuteri-stimulated lymphocytes were sufficient to convey transplantable anti-cancer protection in the cell recipient animals. These data demonstrate that host immune responses to environmental microbes significantly impact and inhibit cancer progression in distal tissues such as mammary glands, even in genetically susceptible mice. This leads us to conclude that consuming fermentative microbes such as L. reuteri may offer a tractable public health approach to help counteract the accumulated dietary and genetic carcinogenic events integral in the Westernized diet and lifestyle. © 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

DOE PAGES

Garbe, James C.; Vrba, Lukas; Sputova, Klara; ...

2014-10-29

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agentsmore » are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.« less

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

PubMed

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells.

PubMed

Kang, Minkyung; Jeong, Wooyoung; Bae, Hyocheol; Lim, Whasun; Bazer, Fuller W; Song, Gwonhwa

2018-03-01

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals. © 2017 Wiley Periodicals, Inc.

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Gas6 Induces Growth, β-Catenin Stabilization, and T-Cell Factor Transcriptional Activation in Contact-Inhibited C57 Mammary Cells

PubMed Central

Goruppi, Sandro; Chiaruttini, Cristina; Ruaro, Maria Elisabetta; Varnum, Brian; Schneider, Claudio

2001-01-01

Gas6 is a growth factor related to protein S that was identified as the ligand for the Axl receptor tyrosine kinase (RTK) family. In this study, we show that Gas6 induces a growth response in a cultured mammalian mammary cell line, C57MG. The presence of Gas6 in the medium induces growth after confluence and similarly causes cell cycle reentry of density-inhibited C57MG cells. We show that Axl RTK but not Rse is efficiently activated by Gas6 in density-inhibited C57MG cells. We have analyzed the signaling required for the Gas6 proliferative effect and found a requirement for PI3K-, S6K-, and Ras-activated pathways. We also demonstrate that Gas6 activates Akt and concomitantly inhibits GSK3 activity in a wortmannin-dependent manner. Interestingly, Gas6 induces up-regulation of cytosolic β-catenin, while membrane-associated β-catenin remains unaffected. Stabilization of β-catenin in C57MG cells is correlated with activation of a T-cell factor (TCF)-responsive transcriptional element. We thus provide evidence that Gas6 is mitogenic and induces β-catenin proto-oncogene stabilization and subsequent TCF/Lef transcriptional activation in a mammary system. These results suggest that Gas6-Axl interaction, through stabilization of β-catenin, may have a role in mammary development and/or be involved in the progression of mammary tumors. PMID:11154277

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells.

PubMed

Yu, Qing Cissy; Song, Wenqian; Lai, Dengwen; Zeng, Yi Arial

2017-08-03

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr + VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

PubMed

Seagroves, Tiffany N; Peacock, Danielle L; Liao, Debbie; Schwab, Luciana P; Krueger, Robin; Handorf, Charles R; Haase, Volker H; Johnson, Randall S

2010-05-01

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

The spectrum of STAT functions in mammary gland development

PubMed Central

Hughes, Katherine; Watson, Christine J.

2012-01-01

The signal transducer and activator of transcription (STAT) family of transcription factors have a spectrum of functions in mammary gland development. In some cases these roles parallel those of STATs in other organ systems, while in other instances the function of individual STATs in the mammary gland is specific to this tissue. In the immune system, STAT6 is associated with differentiation of T helper cells, while in the mammary gland, it has a fundamental role in the commitment of luminal epithelial cells to the alveolar lineage. STAT5A is required for the production of luminal progenitor cells from mammary stem cells and is essential for the differentiation of milk producing alveolar cells during pregnancy. By contrast, the initiation of regression following weaning heralds a dramatic and specific activation of STAT3, reflecting its pivotal role in the regulation of cell death and tissue remodeling during mammary involution. Although it has been demonstrated that STAT1 is regulated during a mammary developmental cycle, it is not yet determined whether it has a specific, non-redundant function. Thus, the mammary gland constitutes an unusual example of an adult organ in which different STATs are sequentially activated to orchestrate the processes of functional differentiation, cell death and tissue remodeling. PMID:24058764

Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro.

PubMed

Mießler, Katharina S; Markov, Alexander G; Amasheh, Salah

2018-01-01

During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation. Copyright © 2017 Elsevier Inc. All rights reserved.

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke

2008-03-21

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation.more » Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.« less

The mammary cellular hierarchy and breast cancer.

PubMed

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

2014-11-01

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 regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.

Biological and genetic properties of the p53 null preneoplastic mammary epithelium

NASA Technical Reports Server (NTRS)

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.

Effects of xanthosine in isoform switch and splice variants of expressed genes in mammary epithelial cells

USDA-ARS?s Scientific Manuscript database

Although intramammary xanthosine (XS) treatment was reported to increase the mammary stem cell population and milk yield in bovine and caprine, underlying molecular mechanisms remain unclear. The goal of this study was to evaluate effects of XS treatment on the mammary transcriptome in early-lactati...

Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas.

PubMed Central

Hand, P H; Thor, A; Wunderlich, D; Muraro, R; Caruso, A; Schlom, J

1984-01-01

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions. Images PMID:6382261

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

PubMed

Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

2017-08-01

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

PubMed

Ferrari, Angelo; Petterino, Claudio; Ratto, Alessandra; Campanella, Chiara; Wurth, Roberto; Thellung, Stefano; Vito, Guendalina; Barbieri, Federica; Florio, Tullio

2012-03-14

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score

Cripto-1 Ablation Disrupts Alveolar Development in the Mouse Mammary Gland through a Progesterone Receptor–Mediated Pathway

PubMed Central

Klauzinska, Malgorzata; McCurdy, David; Rangel, Maria Cristina; Vaidyanath, Arun; Castro, Nadia P.; Shen, Michael M.; Gonzales, Monica; Bertolette, Daniel; Bianco, Caterina; Callahan, Robert; Salomon, David S.; Raafat, Ahmed

2016-01-01

Cripto-1, a member of the epidermal growth factor–Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways. PMID:26429739

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

PubMed

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

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. 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. 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. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development*

PubMed Central

Kitayama, Midori; Mizutani, Kiyohito; Maruoka, Masahiro; Mandai, Kenji; Sakakibara, Shotaro; Ueda, Yuki; Komori, Takahide; Shimono, Yohei; Takai, Yoshimi

2016-01-01

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

Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions

PubMed Central

2010-01-01

Background Breast cancer is the most frequently diagnosed cancer in women. Intraepithelial lesions (IELs), such as usual ductal hyperplasia (UH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors that predict a woman's chance of developing invasive breast cancer. Therefore, a comparative study that establishes an animal model of pre-invasive lesions is needed for the development of preventative measures and effective treatment for both mammary IELs and tumors. The purpose of this study was to characterize the histologic and molecular features of feline mammary IELs and compare them with those in women. Methods Formalin-fixed, paraffin-embedded specimens (n = 205) from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN), and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine (Sassari, Italy). Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were compared to those of humans. Immunohistochemistry for estrogen receptor (ER-alpha), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu) and Ki-67 was performed in IELs and adjacent tumor tissues. Results Intraepithelial lesions were found in 57 of 203 (28%) feline mammary specimens and were categorized as UH (27%), ADH (29%), and DCIS (44%). Most IELs with atypia (ADH and DCIS) were associated with mammary cancer (91%), whereas UH was associated with benign lesions in 53% of cases. Feline IELs were remarkably similar to human IELs. No ER or PR immunoreactivity was detected in intermediate-grade or high-grade DCIS or their associated malignant tumors. HER-2 protein overexpression was found in 27% of IELs. Conclusion The remarkable similarity of feline mammary IELs to those of humans

[NUCLEAR STRUCTURE IN THE SECRETORY CELLS OF MAMMARY GLANDS IN LACTATING AND NON-LACTATING RATS].

PubMed

Tyutina, K V; Skopichev, V G; Bogolyubov, D S; Bogolyubova, I O

2016-01-01

The features of structural and functional organization of the main nuclear compartments and distribution of their key molecular components (chromatin-remodeling protein ATRX, RNA polymerase I and II, and the splicing factor SC35) has been studied in the nuclei of mammary gland cells at different functional states. No significant differences between the nuclei of the cells in the lactating and non-lactating mammary glands have been revealed at the ultrastructural level. At the same time, photometric analysis has revealed higher intensity of nucleoplasmic immunofluorescent staining of mammary glands in the lactating animals when antibodies against the proteins ATRX and SC35 were used. Apparently, this observation reflects the changes of the structural and functional status of chromatin as well as the redistribution of splicing factors between the sites of their deposition and transcription.

GAS6 is an estrogen-inducible gene in mammary epithelial cells

PubMed Central

Mo, Rigen; Zhu, Yiwei Tony; Zhang, Zhongyi; Rao, Sambasiva M.; Zhu, Yi-Jun

2007-01-01

To identify estrogen responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERα interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERα was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells. PMID:17174935

Oncogene-Induced Changes in Mammary Cell Fate and EMT in Breast Tumorigenesis

DTIC Science & Technology

2015-04-01

have worked on the project? Name: Lauren Walheim Project Role: Chemistry pre-medicine undergraduate student Researcher Identifier (e.g. ORCID ID...Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an...Examine if IGF1R alters mammary cell fate in vivo and the effect this has on mammary tumorigenesis (months 13-30) ...…………………………………………….......11 2a

R-spondin3 is associated with basal-progenitor behavior in normal and tumor mammary cells.

PubMed

Tocci, Johanna Melisa; Felcher, Carla María; García Solá, Martín E; Goddio, María Victoria; Zimberlin, María Noel; Rubinstein, Natalia; Srebrow, Anabella; Coso, Omar Adrián; Abba, Martín C; Meiss, Roberto P; Kordon, Edith C

2018-05-10

R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo. Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among breast cancer patients. Thus we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers. Copyright ©2018, American Association for Cancer Research.

A redefinition of the representation of mammary cells and enzyme activities in a lactating dairy cow model.

PubMed

Hanigan, M D; Rius, A G; Kolver, E S; Palliser, C C

2007-08-01

The Molly model predicts various aspects of digestion and metabolism in the cow, including nutrient partitioning between milk and body stores. It has been observed previously that the model underpredicts milk component yield responses to nutrition and consequently overpredicts body energy store responses. In Molly, mammary enzyme activity is represented as an aggregate of mammary cell numbers and activity per cell with minimal endocrine regulation. Work by others suggests that mammary cells can cycle between active and quiescent states in response to various stimuli. Simple models of milk production have demonstrated the utility of this representation when using the model to simulate variable milking and nutrient restriction. It was hypothesized that replacing the current representation of mammary cells and enzyme activity in Molly with a representation of active and quiescent cells and improving the representation of endocrine control of cell activity would improve predictions of milk component yield. The static representation of cell numbers was replaced with a representation of cell growth during gestation and early lactation periods and first-order cell death. Enzyme capacity for fat and protein synthesis was assumed to be proportional to cell numbers. Enzyme capacity for lactose synthesis was represented with the same equation form as for cell numbers. Data used for parameter estimation were collected as part of an extended lactation trial. Cows with North American or New Zealand genotypes were fed 0, 3, or 6 kg of concentrate dry matter daily during a 600-d lactation. The original model had root mean square prediction errors of 17.7, 22.3, and 19.8% for lactose, protein, and fat yield, respectively, as compared with values of 8.3, 9.4, and 11.7% for the revised model, respectively. The original model predicted body weight with an error of 19.7% vs. 5.7% for the revised model. Based on these observations, it was concluded that representing mammary synthetic

Runx2 contributes to the regenerative potential of the mammary epithelium.

PubMed

Ferrari, Nicola; Riggio, Alessandra I; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C; Smalley, Matthew J; Sansom, Owen J; Morris, Joanna; Cameron, Ewan R; Blyth, Karen

2015-10-22

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling.

Runx2 contributes to the regenerative potential of the mammary epithelium

PubMed Central

Ferrari, Nicola; Riggio, Alessandra I.; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C.; Smalley, Matthew J.; Sansom, Owen J.; Morris, Joanna; Cameron, Ewan R.; Blyth, Karen

2015-01-01

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling. PMID:26489514

The Ron Receptor Tyrosine Kinase Negatively Regulates Mammary Gland Branching Morphogenesis

PubMed Central

Meyer, Sara E.; Zinser, Glendon M.; Stuart, William D.; Pathrose, Peterson; Waltz, Susan E.

2009-01-01

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK−/−). Mammary glands from RonTK−/− mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK−/− epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK−/− glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development. PMID:19576199

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

PubMed Central

Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

2013-01-01

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. PMID:24312827

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis

PubMed Central

2012-01-01

Background Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. Results A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

PubMed Central

Mymryk, J S; Berard, D; Hager, G L; Archer, T K

1995-01-01

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin. PMID:7799933

Mouse mammary tumor virus chromatin in human breast cancer cells is constitutively hypersensitive and exhibits steroid hormone-independent loading of transcription factors in vivo.

PubMed

Mymryk, J S; Berard, D; Hager, G L; Archer, T K

1995-01-01

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.

Collision of Ductal Carcinoma In Situ of Anogenital Mammary-like Glands and Vulvar Sarcomatoid Squamous Cell Carcinoma.

PubMed

Tran, Tien A N; Deavers, Michael T; Carlson, J Andrew; Malpica, Anais

2015-09-01

A spectrum of invasive adenocarcinomas presumably arising from the anogenital mammary-like glands of the vulva has been reported. Even rarer are the cases of pure ductal carcinoma in situ that originated from these unique glandular structures. Herein, we report an 81-yr-old woman presented with an invasive well-differentiated squamous cell carcinoma of the vulva. Unexpectedly, the underlying dermis demonstrated a cystically dilated structure that displayed a layer of malignant squamous cells in the periphery, and a second centrally located population of neoplastic cells exhibiting glandular differentiation. In addition, a spindle and pleomorphic malignant cell population consistent with a sarcomatoid carcinoma was identified around the cystic structure. Scattered benign anogenital mammary-like glands were present in the adjacent dermis. The histologic and immunohistochemical findings were consistent with those of vulvar squamous cell carcinoma that has undergone sarcomatoid transformation after spreading in a pagetoid fashion into an underlying focus of ductal carcinoma in situ of anogenital mammary-like gland origin.

Relationship between histology, development and tumorigenesis of mammary gland in female rat

PubMed Central

LÍŠKA, Ján; BRTKO, Július; DUBOVICKÝ, Michal; MACEJOVÁ, Dana; KISSOVÁ, Viktória; POLÁK, Štefan; UJHÁZY, Eduard

2015-01-01

The mammary gland is a dynamic organ that undergoes structural and functional changes associated with growth, reproduction, and post-menopausal regression. The postnatal transformations of the epithelium and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The increased cancer incidence in mammary glands of humans and similarly of rodents in association with their development is believed to be partly explained by proliferative activity together with lesser degree of differentiation, but it is not completely understood how the virgin gland retains its higher susceptibility to carcinogenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer. An early first full-term pregnancy may have a protective effect. Rodent models are useful for investigating potential breast carcinogens. The purpose of this review is to help recognizing histological appearance of the epithelium and the stroma of the normal mammary gland in rats, and throughout its development in relation to tumorigenic potential. PMID:26424555

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

PubMed Central

Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

2011-01-01

Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

The Type 7 Serotonin Receptor, 5-HT7, Is Essential in the Mammary Gland for Regulation of Mammary Epithelial Structure and Function

PubMed Central

Pai, Vaibhav P.; Hernandez, Laura L.; Stull, Malinda A.; Horseman, Nelson D.

2015-01-01

Autocrine-paracrine activity of serotonin (5-hydroxytryptamine, 5-HT) is a crucial homeostatic parameter in mammary gland development during lactation and involution. Published studies suggested that the 5-HT7 receptor type was important for mediating several effects of 5-HT in the mammary epithelium. Here, using 5-HT7 receptor-null (HT7KO) mice we attempt to understand the role of this receptor in mediating 5-HT actions within the mammary gland. We demonstrate for the first time that HT7KO dams are inefficient at sustaining their pups. Histologically, the HT7KO mammary epithelium shows a significant deviation from the normal secretory epithelium in morphological architecture, reduced secretory vesicles, and numerous multinucleated epithelial cells with atypically displaced nuclei, during lactation. Mammary epithelial cells in HT7KO dams also display an inability to transition from lactation to involution as normally seen by transition from a columnar to a squamous cell configuration, along with alveolar cell apoptosis and cell shedding. Our results show that 5-HT7 is required for multiple actions of 5-HT in the mammary glands including core functions that contribute to changes in cell shape and cell turnover, as well as specialized secretory functions. Understanding these actions may provide new interventions to improve lactation performance and treat diseases such as mastitis and breast cancer. PMID:25664318

Effect of DETA-NONOate and papaverine on vasodilation of human internal mammary artery.

PubMed

Rahimi, Nastaran; Dehpour, Ahmad R; Javadi-Paydar, Mehrak; Sohanaki, Hamid; Rabbani, Shahram; Ansari, Mohammad; Tafti, Seyed Hossein Ahmadi

2011-12-01

In this study, the relaxatory effect of DETA-NONOate is compared with that of papaverine on isolated human internal mammary artery. We investigated the inhibitory effects of DETA-NONOate and papaverine on phenylephrine-induced contractile response in internal mammary artery segments. The internal mammary artery segments, taken from methodologically matched patients who underwent coronary artery bypass grafting, were prepared, placed in an organ bath, and contracted with phenylephrine (10(-9) to 10(-4) mol/L) to investigate their relaxatory response to DETA-NONOate or papaverine. Phenylephrine dose-response contraction was obtained after 1, 2, and 3 h in segments pre-incubated with DETA-NONOate or papaverine for 30 min. The EC50 that presented for internal mammary artery segments incubated with DETA-NONOate was 3.523 ± 1.2696 × 10(-7) mol/L, and for papaverine was 3.467 ± 1.2145 × 10(-6) mol/L. In segments pre-incubated with DETA-NONOate, the contractile response to phenylephrine was suppressed in the first 2 h post-incubation, compared with control responsive groups (p < 0.05), but this inhibition was revoked after 3 h post-incubation. We showed that DETA-NONOate has a more significant relaxative effect by comparison with papaverine; moreover, continuous and long-lasting nitric oxide production by DETA-NONOate might be of great importance for the outcome from coronary artery bypass grafting, when internal mammary artery is used as a conduit.

Cripto-1 ablation disrupts alveolar development in the mouse mammary gland through a progesterone receptor-mediated pathway.

PubMed

Klauzinska, Malgorzata; McCurdy, David; Rangel, Maria Cristina; Vaidyanath, Arun; Castro, Nadia P; Shen, Michael M; Gonzales, Monica; Bertolette, Daniel; Bianco, Caterina; Callahan, Robert; Salomon, David S; Raafat, Ahmed

2015-11-01

Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution*

PubMed Central

Lilla, Jennifer N.; Joshi, Ravi V.; Craik, Charles S.; Werb, Zena

2009-01-01

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. PMID:19297327

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

PubMed

Cubeddu, Tiziana; Cacciotto, Carla; Pisanu, Salvatore; Tedde, Vittorio; Alberti, Alberto; Pittau, Marco; Dore, Simone; Cannas, Agnese; Uzzau, Sergio; Rocca, Stefano; Addis, Maria Filippa

2017-07-01

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers. Copyright © 2017 Elsevier B.V. All rights reserved.

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

PubMed Central

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-01-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes. PMID:25688859

Luminal progenitors restrict their lineage potential during mammary gland development.

PubMed

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-02-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

Feline mammary carcinoma stem cells are tumorigenic, radioresistant, chemoresistant and defective in activation of the ATM/p53 DNA damage pathway

PubMed Central

Pang, L.Y.; Blacking, T.M.; Else, R.W.; Sherman, A.; Sang, H.M.; Whitelaw, B.A.; Hupp, T.R.; Argyle, D.J.

2013-01-01

Cancer stem cells were identified in a feline mammary carcinoma cell line by demonstrating expression of CD133 and utilising the tumour sphere assay. A population of cells was identified that had an invasive, mesenchymal phenotype, expressed markers of pluripotency and enhanced tumour formation in the NOD-SCID mouse and chick embryo models. This population of feline mammary carcinoma stem cells was resistant to chemotherapy and radiation, possibly due to aberrant activation of the ATM/p53 DNA damage pathway. Epithelial–mesenchymal transition was a feature of the invasive phenotype. These data demonstrate that cancer stem cells are a feature of mammary cancer in cats. PMID:23219486

Lineage-Restricted Mammary Stem Cells Sustain the Development, Homeostasis, and Regeneration of the Estrogen Receptor Positive Lineage.

PubMed

Van Keymeulen, Alexandra; Fioramonti, Marco; Centonze, Alessia; Bouvencourt, Gaëlle; Achouri, Younes; Blanpain, Cédric

2017-08-15

The mammary gland (MG) is composed of different cell lineages, including the basal and the luminal cells (LCs) that are maintained by distinct stem cell (SC) populations. LCs can be subdivided into estrogen receptor (ER) + and ER - cells. LCs act as the cancer cell of origin in different types of mammary tumors. It remains unclear whether the heterogeneity found in luminal-derived mammary tumors arises from a pre-existing heterogeneity within LCs. To investigate LC heterogeneity, we used lineage tracing to assess whether the ER + lineage is maintained by multipotent SCs or by lineage-restricted SCs. To this end, we generated doxycycline-inducible ER-rtTA mice that allowed us to perform genetic lineage tracing of ER + LCs and study their fate and long-term maintenance. Our results show that ER + cells are maintained by lineage-restricted SCs that exclusively contribute to the expansion of the ER + lineage during puberty and their maintenance during adult life. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Long-term primary culture of mouse mammary tumor cells: production of virus.

PubMed

Young, L J; Cardiff, R D; Ashley, R L

1975-05-01

Long-term primary cultures of mouse mammary tumor cells proved an excellent source of mouse mammary tumor virus (MMTV). Virus purified from these primary cultures had the same morphologic biochemical, immunologic, and biologic characteristics as MMTV. Quantitation of MMTV-protein equivalents released into the medium was measured by the radioimmunoassay for MMTV. Peak production levels were 20-40 mug MMTV protien equivalents/75-cm-2 flask/24 hours. These cultures produced MMTV for as long as 90 days. MMTV cultivation depended on the initial cell-plating density and hormones. Maximal MMTV release was obtained at a plating density of 1 times 10-6 cells/cm-2 in the presence of insulin and hydrocortisone. Insulin alone gave basal levels of MMTV, and hydrocortisone alone increased MMTV release only three-fold, but insulin and hydrocortisone together effected an eightfold increase in MMTV release. This suggested that hydrocortisone had a primary effect on MMTV release and insulin acted synergistically with hydrocortisone to maximize MMTV release.

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin.

PubMed

Calvo, Alfonso; Yokoyama, Yumi; Smith, Lois E; Ali, Iqbal; Shih, Shu-Ching; Feldman, Andrew L; Libutti, Steven K; Sundaram, Ramakrishnan; Green, Jeffrey E

2002-09-20

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth-inhibitory effect on endothelial cell proliferation than wild-type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF(188) mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin-2 mRNA levels are also increased during tumor progression. CD-31 (platelet-endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk-1 and Flt-1, angiopoietin-2, Tie-1, cadherin-5 and PECAM) were significantly decreased in the endostatin-treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model. Copyright 2002 Wiley-Liss, Inc.

Exome-wide Mutation Profile in Benzo[a]pyrene-derived Post-stasis and Immortal Human Mammary Epithelial Cells

PubMed Central

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

2014-01-01

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 towards 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

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

PubMed

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. Copyright © 2014 Elsevier B.V. All rights reserved.

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

DOE PAGES

Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; ...

2014-11-04

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 mutationsmore » 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. In conclusion, 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.« less

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

PubMed

Cereser, Biancastella; Jansen, Marnix; Austin, Emily; Elia, George; McFarlane, Taneisha; van Deurzen, Carolien Hm; Sieuwerts, Anieta M; Daidone, Maria G; Tadrous, Paul J; Wright, Nicholas A; Jones, Louise; McDonald, Stuart Ac

2018-01-01

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival

PubMed Central

2009-01-01

Introduction The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Methods Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. Results In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Conclusions Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.

PubMed

Pai, Vaibhav P; Marshall, Aaron M; Hernandez, Laura L; Buckley, Arthur R; Horseman, Nelson D

2009-01-01

The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

PubMed

Bierie, Brian; Stover, Daniel G; Abel, Ty W; Chytil, Anna; Gorska, Agnieszka E; Aakre, Mary; Forrester, Elizabeth; Yang, Li; Wagner, Kay-Uwe; Moses, Harold L

2008-03-15

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

Bisphenol A Increases Mammary Cancer Risk in Two Distinct Mouse Models of Breast Cancer1

PubMed Central

Weber Lozada, Kristen; Keri, Ruth A.

2011-01-01

Bisphenol A (BPA) is an industrial plasticizer that leaches from food containers during normal usage, leading to human exposure. Early and chronic exposure to endocrine-disrupting environmental contaminants such as BPA elevates the potential for long-term health consequences. We examined the impact of BPA exposure on fetal programming of mammary tumor susceptibility as well as its growth promoting effects on transformed breast cancer cells in vivo. Fetal mice were exposed to 0, 25, or 250 μg/kg BPA by oral gavage of pregnant dams. Offspring were subsequently treated with the known mammary carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). While no significant differences in postnatal mammary development were observed, both low- and high-dose BPA cohorts had a statistically significant increase in susceptibility to DMBA-induced tumors compared to vehicle-treated controls. To determine if BPA also promotes established tumor growth, MCF-7 human breast cancer cells were subcutaneously injected into flanks of ovariectomized NCR nu/nu female mice treated with BPA, 17beta-estradiol, or placebo alone or combined with tamoxifen. Both estradiol- and BPA-treated cohorts formed tumors by 7 wk post-transplantation, while no tumors were detected in the placebo cohort. Tamoxifen reversed the effects of estradiol and BPA. We conclude that BPA may increase mammary tumorigenesis through at least two mechanisms: molecular alteration of fetal glands without associated morphological changes and direct promotion of estrogen-dependent tumor cell growth. Both results indicate that exposure to BPA during various biological states increases the risk of developing mammary cancer in mice. PMID:21636739

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

PubMed Central

Fan, Jie; Cai, Bin; Zeng, Min; Hao, Yanyan

2015-01-01

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium. PMID:21556948

Effect of HSP27 on Human Breast Tumor Cell Growth and Motility

DTIC Science & Technology

1999-08-01

the small stress protein, HSP27 , on growth and motility characteristics of normal and tumor-derived human mammary cell lines. We hypothesized that...cells overexpressing HSP27 would show increased motility, altered chemotactic properties, increased resistance to heat killing and to certain drugs...Donna has prepared and studied 19 clonal MDA23 1 breast tumor cell lines that overexpress human HSP27 , and determined that, while heat resistance is

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells

PubMed Central

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G. E.; Germon, Pierre; Otto, Michael

2016-01-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. PMID:27001539

Characteristics and EGFP expression of goat mammary gland epithelial cells.

PubMed

Zheng, Y-M; He, X-Y; Zhang, Y

2010-12-01

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. © 2010 Blackwell Verlag GmbH.

Mammary gland involution is associated with rapid down regulation of major mammary Ca**2+-ATPases

USDA-ARS?s Scientific Manuscript database

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca**2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca**2+-ATPases and mammary calcium transport is unknown. We found that 24 hours after stopping milk prod...

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential

PubMed Central

Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Zhang, Fuchuang; Gu, Xiang; Wu, Anqi; Wang, Danhan; Chen, Yuanhong; Bandyopadhyay, Abhik; Yeh, I-Tien; Daniel, Benjamin J.; Chen, Yidong; Zou, Yi; Rebel, Vivienne L.; Walter, Christi A.; Lu, Jianxin; Huang, Changjiang; Sun, Lu-Zhe

2016-01-01

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands. PMID:27852980

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential.

PubMed

Dong, Qiaoxiang; Gao, Hui; Shi, Yuanshuo; Zhang, Fuchuang; Gu, Xiang; Wu, Anqi; Wang, Danhan; Chen, Yuanhong; Bandyopadhyay, Abhik; Yeh, I-Tien; Daniel, Benjamin J; Chen, Yidong; Zou, Yi; Rebel, Vivienne L; Walter, Christi A; Lu, Jianxin; Huang, Changjiang; Sun, Lu-Zhe

2016-11-15

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49f hi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49f hi basal-like cells in aged glands.

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

PubMed Central

Young, Christian D.; Lewis, Andrew S.; Rudolph, Michael C.; Ruehle, Marisa D.; Jackman, Matthew R.; Yun, Ui J.; Ilkun, Olesya; Pereira, Renata; Abel, E. Dale; Anderson, Steven M.

2011-01-01

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential. Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1. These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo. PMID:21826239

Effects of xanthosine on gene expression of mammary epithelial cells using RNA sequencing of goat milk fat globules

USDA-ARS?s Scientific Manuscript database

Although intramammary xanthosine (XS) treatment was reported to increase the mammary stem cell population and milk yield in bovine and caprine, underlying molecular mechanisms remain unclear. The goal of this study was to evaluate effects of XS treatment on the mammary transcriptome in early lactati...

Complex mammary carcinoma with metastases to lymph nodes, subcutaneous tissue, and multiple joints in a dog.

PubMed

McCourt, Maggie R; Dieterly, Alexandra M; Mackey, Paige E; Lyon, Shane D; Rizzi, Theresa E; Ritchey, Jerry W

2018-05-07

An 8-year-old, intact female, mixed-breed dog presented to the Oklahoma State University Boren Veterinary Medical Teaching Hospital for evaluation of progressive lameness and joint effusion of multiple joints. Physical examination revealed joint effusion of the elbow, hock, and stifle joints bilaterally, enlarged left axillary and right popliteal lymph nodes, a subcutaneous mass over the left elbow, and a subcutaneous mass involving the left second and third mammary glands. Cytologic examination of the mammary mass, enlarged lymph nodes, and joint fluid from most affected joints revealed a monomorphic population of loosely cohesive neoplastic epithelial cells. The patient was humanely euthanized, and subsequent necropsy with histopathologic examination revealed a complex mammary carcinoma with metastases to enlarged lymph nodes, subcutaneous tissue over the left elbow, and the synovium of multiple joints. Immunohistochemical stains were performed and showed diffusely positive pan cytokeratin, CK8/18, and CK19 staining in the neoplastic luminal epithelial cells of the mammary carcinoma, synovium, and lymph nodes, and showed diffusely positive vimentin staining of the myoepithelial cells. Myoepithelial calponin positivity was diffuse in the mammary mass and lymph nodes but minimal in the synovium. Only the mammary mass showed p63 positivity. Metastatic mammary neoplasia is relatively common in dogs; however, metastasis to the synovium has only been reported once previously in the literature. This is the first case utilizing immunohistochemistry for confirmation and characterization of metastases. © 2018 American Society for Veterinary Clinical Pathology.

Myeloid Cell COX-2 deletion reduces mammary tumor growth through enhanced cytotoxic T-lymphocyte function

PubMed Central

Chen, Edward P.; Markosyan, Nune; Connolly, Emma; Lawson, John A.; Li, Xuanwen; Grant, Gregory R.; Grosser, Tilo; FitzGerald, Garret A.; Smyth, Emer M.

2014-01-01

Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8+ cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8+, but not CD4+ cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8+ CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy. PMID:24590894

p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijjar, Tarlochan; Wigington, Don; Garbe, James C.

1999-08-01

The authors have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMEC). HMEC immortalized following chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined and exhibited slow heterogeneous growth, and contained many non-proliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very graduall2048nverted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerasemore » activity, and stabilization of telomere length. The fully immortal good growing HMEC did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele, and transient expression of the previously imprinted allele. Conditionally immortal 184A1 with mean TRF > 3 kb infected with retroviruses containing the p57 gene exhibited premature slow heterogeneous growth. Conversely, exogenous expression of hTERT, the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMEC which have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57 mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.« less

Expression of truncated Int6/eIF3e in mammary alveolar epithelium leads to persistent hyperplasia and tumorigenesis

PubMed Central

Mack, David L; Boulanger, Corinne A; Callahan, Robert; Smith, Gilbert H

2007-01-01

Introduction Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein. Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas. Methods To validate that Int6sh has transforming activity in vivo, a transgenic mouse model was designed using the whey acidic protein (Wap) promoter to target expression of truncated Int6 to differentiating alveolar epithelial cells in the mammary gland. Microarray analyses were performed on normal, premalignant and malignant WapInt6sh expressing tissues. Results Mammary tumors developed in 42% of WapInt6sh heterozygous parous females at an average age of 18 months. In WapInt6sh mice, the contralateral mammary glands from both tumorous and non-tumorous tissues contained widespread focal alveolar hyperplasia. Only 4% of WapInt6sh non-breeding females developed tumors by 2 years of age. The Wap promoter is active only during estrus in the mammary tissue of cycling non-pregnant mice. Microarray analyses of mammary tissues demonstrated that Int6sh expression in the alveolar tissue altered the mammary transcriptome in a specific manner that was detectable even in the first pregnancy. This Int6sh-specific transcriptome pattern subsequently persisted in both the Int6sh-expressing alveolar hyperplasia and mammary tumors. These observations are consistent with the conclusion that WapInt6sh-expressing alveolar cells survive involution following the cessation of lactation, and subsequently give rise to the mammary tumors that arise in aging multiparous females. Conclusion These observations provide direct in vivo

ATM is required for SOD2 expression and homeostasis within the mammary gland.

PubMed

Dyer, Lisa M; Kepple, Jessica D; Ai, Lingbao; Kim, Wan-Ju; Stanton, Virginia L; Reinhard, Mary K; Backman, Lindsey R F; Streitfeld, W Scott; Babu, Nivetha Ramesh; Treiber, Nicolai; Scharffetter-Kochanek, Karin; McKinnon, Peter J; Brown, Kevin D

2017-12-01

ATM activates the NF-κB transcriptional complex in response to genotoxic and oxidative stress. The purpose of this study was to examine if the NF-κB target gene and critical antioxidant SOD2 (MnSOD) in cultured mammary epithelium is also ATM-dependent, and what phenotypes arise from deletion of ATM and SOD2 within the mammary gland. SOD2 expression was studied in human mammary epithelial cells and MCF10A using RNAi to knockdown ATM or the NF-κB subunit RelA. To study ATM and SOD2 function in mammary glands, mouse lines containing Atm or Sod2 genes containing LoxP sites were mated with mice harboring Cre recombinase under the control of the whey acidic protein promoter. Quantitative PCR was used to measure gene expression, and mammary gland structure was studied using histology. SOD2 expression is ATM- and RelA-dependent, ATM knockdown renders cells sensitive to pro-oxidant exposure, and SOD mimetics partially rescue this sensitivity. Mice with germline deletion of Atm fail to develop mature mammary glands, but using a conditional knockout approach, we determined that Atm deletion significantly diminished the expression of Sod2. We also observed that these mice (termed Atm Δ/Δ ) displayed a progressive lactation defect as judged by reduced pup growth rate, aberrant lobulo-alveolar structure, diminished milk protein gene expression, and increased apoptosis within lactating glands. This phenotype appears to be linked to dysregulated Sod2 expression as mammary gland-specific deletion of Sod2 phenocopies defects observed in Atm Δ/Δ dams. We conclude that ATM is required to promote expression of SOD2 within the mammary epithelium, and that both ATM and SOD2 play a crucial role in mammary gland homeostasis.

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging

PubMed Central

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K.; Wells, Sam; Wikswo, John P.; Zijlstra, Andries; Richmond, Ann

2016-01-01

ABSTRACT We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment. PMID:28243517

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

PubMed

Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K; Wells, Sam; Wikswo, John P; Zijlstra, Andries; Richmond, Ann

2016-01-01

We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

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

PubMed Central

Riskin, Arieh; Mond, Yehudit

2015-01-01

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. PMID:26886772

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

PubMed

Flint, D J; Boutinaud, M; Tonner, E; Wilde, C J; Hurley, W; Accorsi, P A; Kolb, A F; Whitelaw, C B A; Beattie, J; Allan, G J

2005-08-01

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue

Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

PubMed

Chanat, Eric; Le Parc, Annabelle; Lahouassa, Hichem; Badaoui, Bouabid

2016-06-01

In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

Autocrine-paracrine regulation of the mammary gland.

PubMed

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

Misregulation of Stromelysin-1 in Mouse Mammary Tumor Cells Accompanies Acquisition of Stromelysin-1 dependent Invasive Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lochter, A.; Srebrow, A.; Sympson, C.J.

1997-02-21

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. Invasion of Matrigel by tumor cells was largely abolished by metalloproteinasemore » inhibitors, but not by inhibitors of other proteinase families. Inhibition experiments with antisense oligodeoxynucleotides revealed that Matrigel invasion of both cell lines was critically dependent on stromelysin-1 expression. Invasion of collagen, on the other hand, was reduced by only 40-50%. Stromelysin-1 was expressed in both malignant and nonmalignant cells grown on plastic substrata. Its expression was completely inhibited in nonmalignant cells, but up-regulated in tumor cells, in response to Matrigel. Thus misregulation of stromelysin-1 expression appears to be an important aspect of mammary tumor cell progression to an invasive phenotype. The matrix metalloproteinases (MMPs) are a family of extracellular matrix (ECM)-degrading enzymes that have been implicated in a variety of normal developmental and pathological processes, including tumorigenesis. The MMP family comprises at least 15 members with different, albeit overlapping, substrate specificities. During activation of latent MMPs, their propeptides are cleaved and they are converted to a lower molecular weight form by other enzymes, including serine proteinases, and by autocatalytic cleavage. Among the MMPs, stromelysin-1 (SL1) possesses the broadest substrate specificity

ACTIVATION OF DIOXIN RESPONSE ELEMENT (DRE)-ASSOCIATED GENES BY BENZO(A)PYRENE 3,6-QUINONE AND BENZO(A)PYRENE 1,6-QUINONE IN MCF-10A HUMAN MAMMARY EPITHELIAL CELLS

PubMed Central

Burchiel, Scott W.; Thompson, Todd A.; Lauer, Fredine T.; Oprea, Tudor I.

2007-01-01

Benzo(a)pyrene (BaP) is a known human carcinogen and a suspected breast cancer complete carcinogen. BaP is metabolized by several metabolic pathways, some having bioactivation and others detoxification properties. BaP-quinones (BPQs) are formed via cytochrome P450 and peroxidase dependent pathways. Previous studies by our laboratory have shown that BPQs have significant growth promoting and anti-apoptotic activities in human MCF-10A mammary epithelial cells examined in vitro. Previous results suggest that BPQs act via redox-cycling and oxidative stress. However, because two specific BPQs (1,6-BPQ and 3,6-BPQ) differed in their ability to produce reactive oxygen species (ROS) and yet both had strong proliferative and EGF receptor activating activity, we utilized mRNA expression arrays and qRT-PCR to determine potential pathways and mechanisms of gene activation. The results of the present studies demonstrated that 1,6-BPQ and 3,6-BPQ activate dioxin response elements (DRE, also known as xenobiotic response elements, XRE) and anti-oxidant response elements (ARE, also known and electrophile response elements, EpRE). 3,6-BPQ had greater DRE activity than 1,6-BPQ, whereas the opposite was true for the activation of ARE. Both 3,6-BPQ and 1,6-BPQ induced oxidative stress associated genes (HMOX1, GCLC, GCLM, and SLC7A11), phase 2 enzyme genes (NQO1, NQO2, ALDH3A1) PAH metabolizing genes (CYP1B1, EPHX1, AKR1C1), and certain EGF receptor associated genes (EGFR, IER3, ING1, SQSTM1 and TRIM16). The results of these studies demonstrate that BPQs activate numerous pathways in human mammary epithelial cells associated with increased cell growth and survival that may play important roles in tumor promotion. PMID:17466351

Expression of the glutamine metabolism-related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours.

PubMed

Ryu, J-E; Park, H-K; Choi, H-J; Lee, H-B; Lee, H-J; Lee, H; Yu, E-S; Son, W-C

2018-06-01

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism-related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex-type tumours displayed significantly higher GLS1 intensity than simple-type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer. © 2017 John Wiley & Sons Ltd.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

PubMed

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G E; Germon, Pierre; Otto, Michael; Berkova, Nadia

2016-06-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors.

PubMed

Roussos, Evanthia T; Wang, Yarong; Wyckoff, Jeffrey B; Sellers, Rani S; Wang, Weigang; Li, Jiufeng; Pollard, Jeffrey W; Gertler, Frank B; Condeelis, John S

2010-01-01

The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena

Pomegranate exerts chemoprevention of experimentally induced mammary tumorigenesis by suppression of cell proliferation and induction of apoptosis.

PubMed

Bishayee, Anupam; Mandal, Animesh; Bhattacharyya, Piyali; Bhatia, Deepak

2016-01-01

Breast cancer is the second leading cause of cancer-related death in women in the United States and discovery and development of safe chemopreventive drugs is urgently needed. The fruit pomegranate (Punica granatum) is gaining importance because of its various health benefits. This study was initiated to investigate chemopreventive potential of a pomegranate emulsion (PE) against 7,12-dimethylbenz(a)anthracene (DMBA) rat mammary carcinogenesis. The animals were orally administered with PE (0.2-5.0 g/kg), starting 2 wk before and 16 wk following DMBA treatment. PE exhibited a striking reduction of DMBA-induced mammary tumor incidence, total tumor burden, and reversed histopathological changes. PE dose-dependently suppressed cell proliferation and induced apoptosis in mammary tumors. Immunohistochemical studies showed that PE increased intratumor Bax, decreased Bcl2 and manifested a proapoptotic shift in Bax/Bcl2 ratio. In addition, our gene expression study showed PE-mediated upregulation of Bad, caspase-3, caspase-7, caspase-9, poly (ADP ribose) polymerase and cytochrome c in mammary tumors. Thus, PE exerts chemoprevention of mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis mediated through upregulation of Bax and downregulation of Bcl2 in concert with caspase cascades. Pomegranate bioactive phytoconstituents could be developed as a chemopreventive drug to reduce the risk of breast cancer.

MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer.

PubMed

Theodorou, Vassiliki; Kimm, Melanie A; Boer, Mandy; Wessels, Lodewyk; Theelen, Wendy; Jonkers, Jos; Hilkens, John

2007-06-01

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

PubMed

Zhang, Rui; Ma, Huimin; Gao, Yuan; Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial; Liu, Xiaolong; Ge, Gaoxiang

2018-02-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway

PubMed Central

Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial

2018-01-01

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation. PMID:29420538

Mammary Cancer and Activation of Transposable Elements

DTIC Science & Technology

2015-03-01

EGFP). No other cell type in the mammary fat pad was observed to express EGFP. Wholemount and FACS analyses of mammary fat pads after involution from...were sacrificed for PI-MEC isolation in groups of up to 4 control or cancer-prone uniparous or triparous females. Both 4 th mammary fat pads were...to unknown reason (n=46), and smaller numbers of animals with various conditions (malocclusion, head tilt , dystocia, respiratory complaints, identity

Growth Hormone and Insulin-Like Growth Factor-I in the Transition from Normal Mammary Development to Preneoplastic Mammary Lesions

PubMed Central

Kleinberg, David L.; Wood, Teresa L.; Furth, Priscilla A.; Lee, Adrian V.

2009-01-01

Adult female mammary development starts at puberty and is controlled by tightly regulated cross-talk between a group of hormones and growth factors. Although estrogen is the initial driving force and is joined by luteal phase progesterone, both of these hormones require GH-induced IGF-I in the mammary gland in order to act. The same group of hormones, when experimentally perturbed, can lead to development of hyperplastic lesions and increase the chances, or be precursors, of mammary carcinoma. For example, systemic administration of GH or IGF-I causes mammary hyperplasia, and overproduction of IGF-I in transgenic animals can cause the development of usual or atypical hyperplasias and sometimes carcinoma. Although studies have clearly demonstrated the transforming potential of both GH and IGF-I receptor in cell culture and in animals, debate remains as to whether their main role is actually instructive or permissive in progression to cancer in vivo. Genetic imprinting has been shown to occur in precursor lesions as early as atypical hyperplasia in women. Thus, the concept of progression from normal development to cancer through precursor lesions sensitive to hormones and growth factors discussed above is gaining support in humans as well as in animal models. Indeed, elevation of estrogen receptor, GH, IGF-I, and IGF-I receptor during progression suggests a role for these pathways in this process. New agents targeting the GH/IGF-I axis may provide a novel means to block formation and progression of precursor lesions to overt carcinoma. A novel somatostatin analog has recently been shown to prevent mammary development in rats via targeted IGF-I action inhibition at the mammary gland. Similarly, pegvisomant, a GH antagonist, and other IGF-I antagonists such as IGF binding proteins 1 and 5 also block mammary gland development. It is, therefore, possible that inhibition of IGF-I action, or perhaps GH, in the mammary gland may eventually play a role in breast cancer

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT.

PubMed

Haga, Kei; Ohno, Shin-ichi; Yugawa, Takashi; Narisawa-Saito, Mako; Fujita, Masatoshi; Sakamoto, Michiie; Galloway, Denise A; Kiyono, Tohru

2007-02-01

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.

2008-11-14

REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

Mammary candidiasis: molecular-based detection of Candida species in human milk samples.

PubMed

Mutschlechner, W; Karall, D; Hartmann, C; Streiter, B; Baumgartner-Sigl, S; Orth-Höller, D; Lass-Flörl, C

2016-08-01

In this prospective and monocentric study, we investigated the performance of a commercialized real-time polymerase chain reaction (RT-PCR) test system for the specific detection of DNA from Candida albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis in human milk samples of patients suspicious of mammary candidiasis. For this purpose, 43 breast-feeding women with characteristic symptoms of mammary candidiasis and 40 asymptomatic controls were enrolled. By culture, Candida spp. were detected in 8.8 % (4/46) and 9.3 % (4/43) of patient and control samples, respectively. Candida albicans (2/46), C. parapsilosis (1/46), and C. guilliermondii (1/46) were present in patient samples, and C. lusitaniae (3/43) and C. guilliermondii (1/43) were present in the controls. After RT-PCR was applied, Candida spp. were found to be present in 67.4 % (31/46) and 79.1 % (34/43) of patient and control samples investigated, respectively. PCR detection of C. albicans and C. parapsilosis revealed only a low sensitivity and specificity of 67.4 % and 41.9 %, respectively. Our data do not support the use of Candida RT-PCR for sensitive and specific diagnosis of mammary candidiasis.

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

PubMed Central

Shahid, Muhammad; Wang, Jianfang; Gu, Xiaolong; Chen, Wei; Ali, Tariq; Gao, Jian; Han, Dandan; Yang, Rui; Fanning, Séamus; Han, Bo

2017-01-01

Prototheca zopfii infections are becoming global concerns in humans and animals. Bovine protothecal mastitis is characterized by deteriorating milk quality and quantity, thus imparting huge economic losses to dairy industry. Previous published studies mostly focused on the prevalence and characterization of P. zopfii from mastitis. However, the ultrastructural pathomorphological changes associated with apoptosis in bovine mammary epithelial cells (bMECs) are not studied yet. Therefore, in this study we aimed to evaluate the in vitro comparative apoptotic potential of P. zopfii genotype-I and -II on bMECs using flow cytometry, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results showed fast growth rate and higher adhesion capability of genotype-II in bMECs as compared with genotype-I. The viability of bMECs infected with P. zopfii genotype-II was significantly decreased after 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control cells. Contrary, genotype-I couldn't show any significant effects on cell viability. Moreover, after infection of bMECs with genotype-II, the apoptosis increased significantly at 12 h (p < 0.05) and 24 h (p < 0.01) as compared with control group. Genotype-I couldn't display any significant effects on cell apoptosis. The host specificity of P. zopfii was also tested in mouse osteoblast cells, and the results suggest that genotype-I and -II could not cause any significant apoptosis in these cell lines. SEM interpreted the pathomorphological alterations in bMECs after infection. Adhesion of P. zopfii with cells and further disruption of cytomembrane validated the apoptosis caused by genotype-II under SEM. While genotype-1 couldn't cause any significant apoptosis in bMECs. Furthermore, genotype-II induced apoptotic manifested specific ultrastructure features, like cytoplasmic cavitation, swollen mitochondria, pyknosis, cytomembrane disruption, and appearance of apoptotic bodies under TEM. The

Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium

PubMed Central

2013-01-01

Introduction Early pregnancy has a strong protective effect against breast cancer in humans and rodents, but the underlying mechanism is unknown. Because breast cancers are thought to arise from specific cell subpopulations of mammary epithelia, we studied the effect of parity on the transcriptome and the differentiation/proliferation potential of specific luminal and basal mammary cells in mice. Methods Mammary epithelial cell subpopulations (luminal Sca1-, luminal Sca1+, basal stem/progenitor, and basal myoepithelial cells) were isolated by flow cytometry from parous and age-matched virgin mice and examined by using a combination of unbiased genomics, bioinformatics, in vitro colony formation, and in vivo limiting dilution transplantation assays. Specific findings were further investigated with immunohistochemistry in entire glands of parous and age-matched virgin mice. Results Transcriptome analysis revealed an upregulation of differentiation genes and a marked decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells of parous mice. Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3. This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo. As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells. Because recombinant Wnt4 rescued the proliferation defect of basal stem/progenitor cells in vitro, reduced Wnt4 secretion appears to be causally related to parity-induced alterations of basal stem

Mammary Tumors Initiated by Constitutive Cdk2 Activation Contain an Invasive Basal-like Component1

PubMed Central

Corsino, Patrick E; Davis, Bradley J; Nörgaard, Peter H; Teoh Parker, Nicole N; Law, Mary; Dunn, William; Law, Brian K

2008-01-01

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, α smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and β-catenin and p120ctn localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, β-catenin, and p120ctn to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers. PMID:18953433

Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis

PubMed Central

Williams, Terence M.; Sotgia, Federica; Lee, Hyangkyu; Hassan, Ghada; Di Vizio, Dolores; Bonuccelli, Gloria; Capozza, Franco; Mercier, Isabelle; Rui, Hallgeir; Pestell, Richard G.; Lisanti, Michael P.

2006-01-01

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1−/− mice and performed a series of mammary transplant studies, using both wild-type and Cav-1−/− mammary fat pads. Cav-1−/− mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1−/− mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis. PMID:17071600

Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.

PubMed

Derksen, Patrick W B; Braumuller, Tanya M; van der Burg, Eline; Hornsveld, Marten; Mesman, Elly; Wesseling, Jelle; Krimpenfort, Paul; Jonkers, Jos

2011-05-01

Breast cancer is the most common malignancy in women of the Western world. Even though a large percentage of breast cancer patients show pathological complete remission after standard treatment regimes, approximately 30-40% are non-responsive and ultimately develop metastatic disease. To generate a good preclinical model of invasive breast cancer, we have taken a tissue-specific approach to somatically inactivate p53 and E-cadherin, the cardinal cell-cell adhesion receptor that is strongly associated with tumor invasiveness. In breast cancer, E-cadherin is found mutated or otherwise functionally silenced in invasive lobular carcinoma (ILC), which accounts for 10-15% of all breast cancers. We show that mammary-specific stochastic inactivation of conditional E-cadherin and p53 results in impaired mammary gland function during pregnancy through the induction of anoikis resistance of mammary epithelium, resulting in loss of epithelial organization and a dysfunctional mammary gland. Moreover, combined inactivation of E-cadherin and p53 induced lactation-independent development of invasive and metastatic mammary carcinomas, which showed strong resemblance to human pleomorphic ILC. Dissemination patterns of mouse ILC mimic the human malignancy, showing metastasis to the gastrointestinal tract, peritoneum, lung, lymph nodes and bone. Our results confirm that loss of E-cadherin contributes to both mammary tumor initiation and metastasis, and establish a preclinical mouse model of human ILC that can be used for the development of novel intervention strategies to treat invasive breast cancer.

Mammary and extramammary Paget's disease

PubMed Central

Lloyd, J; Flanagan, A

2000-01-01

Mammary and extramammary Paget's disease are uncommon intraepithelial adenocarcinomas. Both conditions have similar clinical features, which mimic inflammatory and infective diseases. Histological diagnostic confusion can arise between Paget's disease and other neoplastic conditions affecting the skin, with the most common differential diagnoses being malignant melanoma and atypical squamous disease. The glandular differentiation of both mammary Paget's disease and extramammary Paget's disease is indicated by morphological appearances, the presence of intracellular mucin in many cases, and positive immunohistochemical staining for glandular cytokeratins, epithelial membrane antigen, and carcinoembryonic antigen. This article provides an overview of mammary and extramammary Paget's disease and discusses recent evidence regarding the cell of origin. The concepts of primary and secondary Paget's disease are presented and the differential diagnosis is discussed with reference to immunohistochemical markers that might be of diagnostic value. Key Words: mammary Paget's disease • extramammary Paget's disease PMID:11064666

Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche.

PubMed

Chakrabarti, Rumela; Celià-Terrassa, Toni; Kumar, Sushil; Hang, Xiang; Wei, Yong; Choudhury, Abrar; Hwang, Julie; Peng, Jia; Nixon, Briana; Grady, John J; DeCoste, Christina; Gao, Jie; van Es, Johan H; Li, Ming O; Aifantis, Iannis; Clevers, Hans; Kang, Yibin

2018-05-17

The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. Here, we show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feed back loop that promotes the function of Dll1 + MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1/Notch-mediated signaling. Copyright © 2018, American Association for the Advancement of Science.

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

PubMed

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 (<3% milk protein; <3.5% 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. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Influence of tangeretin on tamoxifen's therapeutic benefit in mammary cancer.