Science.gov

Sample records for human mammary cell

  1. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

  2. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    robust and reproducible methodology to detect, quantify and isolate stem cells in normal human mammary tissue, using a xenotransplantation system...covers the first year of the grant, during which substantial progress has been made in the development and validation of the xenotransplantation assay...Subrenal xenotransplantation surgery. The hair on the back of anesthetized mice was shaved, and the skin swabbed with 70% alcohol. An anterior to

  3. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  4. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2008-10-01

    9 Appendix……………………………………………………………………………… 10 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A...Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turashvili, Samuel Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human...Eirew, Afshin Raouf, John Stingl, Gulisa Turashvili, Allen Delaney, Joanne Emerman, Marco Marra and Samuel Aparicio . “Stem Cells in the Mammary Gland

  5. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  6. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    Immortal Transformation of Cultured Human Mammary Epithelial Cells. Cellular Oncology, 26:248-251, 2004. Rodier , F., Kim, S-H., Nijjar, T., Yaswen, P...Promoter, Mol. Cell Biol.: 25:3923-3933, 2005. Goldstein, J, Rodier , F, Garbe, J, Stampfer, M, Campisi, J, Caspase-independent cytochrome c release is a

  7. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  8. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  9. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

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

    PubMed

    Adkins, Y; Lönnerdal, B

    2001-01-01

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

  11. Humanization of the mouse mammary gland.

    PubMed

    Wronski, A; Arendt, L M; Kuperwasser, Charlotte

    2015-01-01

    Although mouse models have provided invaluable information on the mechanisms of mammary gland development, anatomical and developmental differences between human and mice limit full understanding of this fundamental process. Humanization of the mouse mammary gland by injecting immortalized human breast stromal cells into the cleared murine mammary fat pad enables the growth and development of human mammary epithelial cells or tissue. This facilitates the characterization of human mammary gland development or tumorigenesis by utilizing the mouse mammary fat pad. Here we describe the process of isolating human mammary stromal and epithelial cells as well as their introduction into the mammary fat pads of immunocompromised mice.

  12. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  13. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    NASA Astrophysics Data System (ADS)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  14. Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

    PubMed Central

    Martignani, Eugenio; Eirew, Peter; Accornero, Paolo

    2010-01-01

    Background In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. Methods and Findings We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. Conclusions These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial

  15. Transcriptome analysis of the normal human mammary cell commitment and differentiation process.

    PubMed

    Raouf, Afshin; Zhao, Yun; To, Karen; Stingl, John; Delaney, Allen; Barbara, Mary; Iscove, Norman; Jones, Steven; McKinney, Steven; Emerman, Joanne; Aparicio, Samuel; Marra, Marco; Eaves, Connie

    2008-07-03

    Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process, but the molecular mechanisms involved, particularly in the human mammary gland, are poorly understood. To address this issue, we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified, and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together, these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation.

  16. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  17. 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.

  18. 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.

  19. Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and...

  20. 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

    sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene expression profiles of...AD_____________ Award Number: W81XWH-12-1-0106 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making Improve

  1. 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

    Conclusion. We have used FAC sorting data coming from human and mouse adult mammary gland , and coming from the fetal mammary rudiment, to define gene...AD_____________ Award Number: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human...4. TITLE AND SUBTITLE Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic

  2. Genes involved in immortalization of human mammary cells

    SciTech Connect

    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 of 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 enforcing a Raf

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

    SciTech Connect

    Twite, Nicolas; Andrei, Graciela; Kummert, Caroline; Donner, Catherine; Perez-Morga, David; De Vos, Rita; Snoeck, Robert; Marchant, Arnaud

    2014-07-15

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

  4. Effect of Estrogen on Mutagenesis in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2005-06-01

    estrogen signaling through the estrogen receptor alpha (ERa) may induce a mutator phenotype by suppressing DNA repair activity in ERa positive mammary...measure DNA MMR activity in live cells. We have also developed a method to measure mutation rate as a function of estrogen/ER signaling . Our goals were to...whether 1713-estradiol signaling through ER inhibits DNA MMR activity, we developed a method that can quantitatively assess MMR efficiency in live

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

    SciTech Connect

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

    1999-02-01

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

  6. Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Craise, L. M.; Durante, M.

    1997-01-01

    Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

  7. Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells.

    PubMed

    Montales, Maria Theresa E; Rahal, Omar M; Nakatani, Hajime; Matsuda, Tsukasa; Simmen, Rosalia C M

    2013-07-01

    Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GEN's role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GEN's effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, and Pparγ (Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor β (Erβ (Esr2)) gene expression and was modestly recapitulated by ERβ-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction of Erβ expression by siRNA targeting increased Pparγ transcript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.

  8. Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids.

    PubMed

    Qu, Ying; Han, Bingchen; Gao, Bowen; Bose, Shikha; Gong, Yiping; Wawrowsky, Kolja; Giuliano, Armando E; Sareen, Dhruv; Cui, Xiaojiang

    2017-02-14

    Human induced pluripotent stem cells (iPSCs) can give rise to multiple cell types and hold great promise in regenerative medicine and disease-modeling applications. We have developed a reliable two-step protocol to generate human mammary-like organoids from iPSCs. Non-neural ectoderm-cell-containing spheres, referred to as mEBs, were first differentiated and enriched from iPSCs using MammoCult medium. Gene expression profile analysis suggested that mammary gland function-associated signaling pathways were hallmarks of 10-day differentiated mEBs. We then generated mammary-like organoids from 10-day mEBs using 3D floating mixed gel culture and a three-stage differentiation procedure. These organoids expressed common breast tissue, luminal, and basal markers, including estrogen receptor, and could be induced to produce milk protein. These results demonstrate that human iPSCs can be directed in vitro toward mammary lineage differentiation. Our findings provide an iPSC-based model for studying regulation of normal mammary cell fate and function as well as breast disease development.

  9. Human Mammary Epithelial Cell Transformation by Rho GTPase through a Novel Mechanism

    DTIC Science & Technology

    2008-08-01

    cells lining the milk -forming ducts of the mammary gland (for review [2]). Deliberate transformation of these cells provides a practical window into human...from milk do not exhibit spontaneous immortalization and thus provide suitable models of human cell transformation. Immortalization of HMECs in culture...immortalize fully a large proportion of preselection HMECs [8]. Human milk is an easily available source of relatively pure HMECs that are thought to be

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

    PubMed

    Bussard, Karen M; Smith, Gilbert H

    2012-01-01

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

  11. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    DTIC Science & Technology

    2012-04-01

    diverse transformed HMEC lines with defined genetic alterations may aid the identification of potential therapeutic treatments , including...human model systems to test potential therapeutics, could facilitate individualized treatment and possibly prevention. The main variables thought to...epithelial cells. Middle, corresponding cell culture models used in this study. Red, treatment or genetic manipulation used. Cell models are described in

  12. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments

    SciTech Connect

    LaBarge, Mark A; Nelson, Celeste M; Villadsen, Rene; Fridriksdottir, Agla; Ruth, Jason R; Stampfer, Martha R; Petersen, Ole W; Bissell, Mina J

    2008-09-19

    In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages.

  13. Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment

    PubMed Central

    2011-01-01

    Introduction The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised. Methods Here, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made. Results Several of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species. Conclusions These results provide new insights into genetic regulatory mechanisms of mammary development, particularly

  14. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments†

    PubMed Central

    Nelson, Celeste M.; Villadsen, Rene; Fridriksdottir, Agla; Ruth, Jason R.; Stampfer, Martha R.; Petersen, Ole W.

    2010-01-01

    In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell–cell versus cell–ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages. PMID:20023793

  15. Differentiation and Genomic Instability in a Human Mammary Cell Model

    NASA Technical Reports Server (NTRS)

    Richmond, R.; Kale, R.; Pettengill, O.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

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

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

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

  17. 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).

  18. Gene Regulation by Retinoid Receptors in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2002-10-01

    altered response to rECM, ECM sig- (919) 668-2458. E-mail: seewa001@mcAuke.edu naling pathways may utilize tumor suppressor checkpoints *Abbreviations...1998). In the tumor suppressor p53 in ECM-induced growth arrest, po- normal mammary gland, the x3/13 1 integrin is expressed at larity, and apoptosis...as a tumor mune IgG and grown in rECM. Electron micrographs of suppressor and in promoting differentiation, little is known early passage p53- HMEC-E6

  19. Prevention of Human Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-07-01

    of HER-2/neu oncogene-transformed human mammary epithelial cells by a green tea polyphenol. Breast Cancer Res Treat 38:100; 1996 8. Telang NT, Katdare...apoptosis and anchorage-dependent colony forming efficiency. Appendix Figure 5 (AF-5): Effect of (-) epigallo catechin gallate (EGCG) on 184- B5/HER cells is... tea polyphenol. Ann. NY Acad. Sci. 768: 215- 222, 1995. Appendix Pubilcation 5 (AP-5): Fishman J, Osborne MP, Telang NT. The role of estrogen in

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

    SciTech Connect

    Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun; Yarishkin, Oleg V.; Bae, Young Min; Kim, Jae Gon; O'Grady, Scott M.; Kang, Kyung-Sun; Ryu, Pan Dong; Lee, So Yeong

    2009-06-26

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

  1. Distinct Effects of EGFR Ligands on Human Mammary Epithelial Cell Differentiation

    PubMed Central

    Mukhopadhyay, Chandrani; Zhao, Xiangshan; Maroni, Dulce; Band, Vimla; Naramura, Mayumi

    2013-01-01

    Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5+K19- hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5+K19- hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5+K19- hMEC differentiation. The present data validate the utility of the K5+K19- hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation. PMID:24124521

  2. Distinct effects of EGFR ligands on human mammary epithelial cell differentiation.

    PubMed

    Mukhopadhyay, Chandrani; Zhao, Xiangshan; Maroni, Dulce; Band, Vimla; Naramura, Mayumi

    2013-01-01

    Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+)K19(-) hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+)K19(-) hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5(+)K19(-) hMEC differentiation. The present data validate the utility of the K5(+)K19(-) hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.

  3. 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

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

    PubMed

    Teoh-Fitzgerald, M L; Fitzgerald, M P; Zhong, W; Askeland, R W; Domann, F E

    2014-01-16

    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

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

    PubMed Central

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

    1994-01-01

    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 deletion 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 G1 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. Images PMID:7511207

  6. 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.

  7. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    DTIC Science & Technology

    2008-10-01

    using the LBNL HTA µarray core facility. These results are consistent with the immunologic data, and also indicate that the milk-derived cells...grown in a lower stress medium were more vulnerable to c-myc immortalization and telomerase upregulation. Indeed, early passage pre-stasis 184 HMEC... early and late passage cultures of 184 and 48R HMEC will be transduced first with p16sh, and then c-myc, to determine if cells that are closer to

  8. 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.

  9. 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).

  10. Microsatellite instability in human mammary epithelial cells transformed by heavy ions

    NASA Astrophysics Data System (ADS)

    Yanada, S.; Yang, T. C.; George, K.; Okayasu, R.; Ando, K.; Tsujii, H.

    1998-11-01

    We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CY17 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes, 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.

  11. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    SciTech Connect

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    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 linked 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.

  12. 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

  13. 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.

  14. 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).

  15. Genotoxic effects of five polycyclic aromatic hydrocarbons in human and rat mammary epithelial cells

    SciTech Connect

    Mane, S.S.; Purnell, D.M.; Hsu, Ih-chang )

    1990-01-01

    Five polycyclic aromatic hydrocarbons (PAHs) of different carcinogenic activities were evaluated for their effects on DNA synthesis ({sup 3}HTdR labeling index (L.I.)) of rat and human mammary epithelial cells (MEC) and for their effects on chromosomes in MEC-mediated sister chromatid exchange (SCE) assays. When compared with DMSO-treated cells, exposures of rat MEC to the two most potent carcinogens, i.e., 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P), resulted in a 45-62% reduction in the L.I. of rat MEC. Another carcinogen, 20-methylcholanthrene (MCA), produced a 35-48% reduction in L.I., while the noncarcinogenic PAHs, 1,2-benzanthracene (BA) and benzo(e)pyrene (B(e)P), showed no effect. Similarly, exposures of human MEC to DMBA and B(a)P resulted in a 50-90% depression in L.I. while BA was significantly less effective. When co-cultivated with Chinese hamster V-79 cells in the presence of PAH, both rat and human MEC can activate and release the active metabolites to induce SCE in V-79 cells. Comparing depression of L.I., SCE, and in vivo carcinogenicity for the 5 PAHs, SCE mediated by rat MEC is better correlated with carcinogenicity in rat than L.I. depression.

  16. 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

  17. Whey acidic protein (WAP) depresses the proliferation of mouse (MMT) and human (MCF-7) mammary tumor cells.

    PubMed

    Nukumi, Naoko; Iwamori, Tokuko; Naito, Kunihiko; Tojo, Hideaki

    2005-10-01

    We previously reported that the enforced expression of exogenous whey acidic protein (WAP) significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 and EpH4/H6 cells). This paper presents the first evidence that WAP also depresses the proliferation of mammary tumor cells from mouse (MMT cells) and human (MCF-7 cells). We established WAP-clonal MMT and MCF-7 cell lines, and confirmed the secretion of WAP from the WAP-clonal cells into culture medium. The enforced expression of WAP significantly inhibited the proliferation of MMT and MCF-7 cells in in vitro culture. FACScan analyses revealed that G0/G1 phase cell-cycle progression was disordered and elongated in the WAP-clonal MMT and MCF-7 cells compared to that of the control cells. The expression of cyclin D1 was significantly decreased in the WAP-clonal MMT and MCF-7 cells, suggesting that progression from the G1 to the S phase was delayed in the WAP-clonal cells. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary tumor cells via a paracrine mechanism.

  18. 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.

  19. X-ray radiation-induced effects in human mammary epithelial cells investigated by Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Risi, R.; Manti, L.; Perna, G.; Lasalvia, M.; Capozzi, V.; Delfino, I.; Lepore, M.

    2012-06-01

    Micro-Raman technique can be particularly useful to investigate the chemical changes induced in structure, protein, nucleic acid, lipid, and carbohydrate contents of cells. The aim of this work is to inspect the possibility to employ Raman microspectroscopy to detect biochemical modifications in human mammary epithelial cells after exposure to different Xray doses. The samples consisted of cells cultured on polylysine-coated glass coverslips. After the exposition, control and treated cells were washed in phosphate-buffered saline (PBS) and then fixed in paraformaldehyde 3.7%. They were examined using a confocal micro-Raman system equipped with a He-Ne laser (λ = 632.8 nm; power on the sample= 3.5mW). Differences in the intensity ratio of specific Raman vibrational markers commonly assigned to phenylalanine and tyrosine amino acids (at 1000, 1030, 1618 cm-1), DNA bases (787, 1090, 1305 cm-1), and amide III (1237, and 1265 cm-1) with respect a reference peak (the one of lipids at 1450 cm-1) were evidenced between control and exposed cells. These differences may be indicative of damage in exposed cells as the fragmentation of individual amino acids and DNA bases, crosslink effects in molecular structure of DNA and protein conformational change that especially tend to "unwind" the protein due to the breaking of hydrogen bonds between peptide chains.

  20. 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.

  1. 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

  2. Cloning of Mammary Stem Cells

    DTIC Science & Technology

    2001-11-01

    these parity-induced cells do represent a totipotent mammary stem cell population per se, but these cells might support stem cell maintenance as... Stem Cells PRINCIPAL INVESTIGATOR: Dr. Kay-Uwe Wagner CONTRACTING ORGANIZATION: University of Nebraska Medical Center Omaha, Nebraska 68198-6810 REPORT...Mammary Stem Cells DAMD17-00-1-0641 6. AUTHOR(S) Dr. Kay-Uwe Wagner 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT

  3. Estrogen receptor alpha inhibits senescence-like phenotype and facilitates transformation induced by oncogenic ras in human mammary epithelial cells

    PubMed Central

    Liu, Zhao; Wang, Long; Yang, Junhua; Bandyopadhyay, Abhik; Kaklamani, Virginia; Wang, Shui; Sun, Lu-Zhe

    2016-01-01

    Exposure to estrogen has long been associated with an increased risk of developing breast cancer. However, how estrogen signaling promotes breast carcinogenesis remains elusive. Senescence is known as an important protective response to oncogenic events. We aimed to elucidate the role of estrogen receptor alpha (ERα) on senescence in transformed human mammary epithelial cells and breast cancer cells. Our results show that ectopic expression of oncoprotein H-ras-V12 in immortalized human mammary epithelial cells (HMEC) significantly inhibited the phosphorylation of the retinoblastoma protein (Rb) and increased the activity of the senescence-associated beta-galactosidase (SA-β-Gal). These senescence-like phenotypes were reversed by ectopic expression of ERα. Similar inhibition of the H-ras-V12-induced SA-β-Gal activity by ERα was also observed in the human mammary epithelial MCF-10A cells. Co-expression of ERα and H-ras-V12 resulted in HMEC anchorage-independent growth in vitro and tumor formation in vivo. Furthermore, inhibition of ERα expression induced senescence-like phenotypes in ERα positive human breast cancer cells such as increased activity of SA-β-Gal, decreased phosphorylation of RB, and loss of mitogenic activity. Thus, the suppression of cellular senescence induced by oncogenic signals may be a major mechanism by which ERα promotes breast carcinogenesis. PMID:27259243

  4. Stress signaling from human mammary epithelial cells contributes to phenotypes of mammographic density.

    PubMed

    DeFilippis, Rosa Anna; Fordyce, Colleen; Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D

    2014-09-15

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal nontumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared with epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g., activin A and CD36) to prevent breast cancer.

  5. Stress Signaling from Human Mammary Epithelial Cells Contributes to Phenotypes of Mammographic Density

    PubMed Central

    Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V.; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D.

    2014-01-01

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal non-tumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared to epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g. activin A, CD36) to prevent breast cancer. PMID:25172842

  6. Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

    PubMed Central

    Devailly, Guillaume; Grandin, Mélodie; Perriaud, Laury; Mathot, Pauline; Delcros, Jean-Guy; Bidet, Yannick; Morel, Anne-Pierre; Bignon, Jean-Yves; Puisieux, Alain; Mehlen, Patrick; Dante, Robert

    2015-01-01

    DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells. PMID:26007656

  7. Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells.

    PubMed

    Devailly, Guillaume; Grandin, Mélodie; Perriaud, Laury; Mathot, Pauline; Delcros, Jean-Guy; Bidet, Yannick; Morel, Anne-Pierre; Bignon, Jean-Yves; Puisieux, Alain; Mehlen, Patrick; Dante, Robert

    2015-07-13

    DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.

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

    PubMed Central

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

    2004-01-01

    Several different transcription factors, including estrogen receptor, progesterone receptor, and ETS family members, have been implicated in human breast cancer, indicating that transcription factor-induced alterations in gene expression underlie mammary cell transformation. ESE-1 is an epithelium-specific ETS transcription factor that contains two distinguishing domains, a serine- and aspartic acid-rich (SAR) domain and an AT hook domain. ESE-1 is abundantly expressed in human breast cancer and trans-activates epithelium-specific gene promoters in transient transfection assays. While it has been presumed that ETS factors transform mammary epithelial cells via their nuclear transcriptional functions, here we show (i) that ESE-1 protein is cytoplasmic in human breast cancer cells; (ii) that stably expressed green fluorescent protein-ESE-1 transforms MCF-12A human mammary epithelial cells; and (iii) that the ESE-1 SAR domain, acting in the cytoplasm, is necessary and sufficient to mediate this transformation. Deletion of transcriptional regulatory or nuclear localization domains does not impair ESE-1-mediated transformation, whereas fusing the simian virus 40 T-antigen nuclear localization signal to various ESE-1 constructs, including the SAR domain alone, inhibits their transforming capacity. Finally, we show that the nuclear localization of ESE-1 protein induces apoptosis in nontransformed mammary epithelial cells via a transcription-dependent mechanism. Together, our studies reveal two distinct ESE-1 functions, apoptosis and transformation, where the ESE-1 transcription activation domain contributes to apoptosis and the SAR domain mediates transformation via a novel nonnuclear, nontranscriptional mechanism. These studies not only describe a unique ETS factor transformation mechanism but also establish a new paradigm for cell transformation in general. PMID:15169914

  9. Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation

    PubMed Central

    Sokol, Ethan S.; Miller, Daniel H.; Mathis, Robert A.; Gupta, Piyush B.

    2015-01-01

    The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available. PMID:25894653

  10. Kefir extracts suppress in vitro proliferation of estrogen-dependent human breast cancer cells but not normal mammary epithelial cells.

    PubMed

    Chen, Chujian; Chan, Hing Man; Kubow, Stan

    2007-09-01

    Anti-tumorigenic effects have been demonstrated in animal studies from the intake of kefir, a traditional fermented milk product believed to originate from the Caucasian mountains of Russia. In the present study, the antiproliferative effects of extracts of kefir, yogurt, and pasteurized cow's milk on human mammary cancer cells (MCF-7) and normal human mammary epithelial cells (HMECs) was investigated at doses of 0.31%, 0.63%, 1.25%, 2.5%, 5%, and 10% (vol/vol). After 6 days of culture, extracts of kefir-fermented milk depressed MCF-7 cell growth in a dose-dependent manner, showing 29% inhibition of proliferation at a concentration as low as 0.63%, whereas yogurt extracts began to show dose-dependent antiproliferative effects only at the 2.5% dose. Moreover, at the 2.5% dose, kefir extracts decreased the MCF-7 cell numbers by 56%, while yogurt extracts decreased MCF-7 cell proliferation by only 14%. No antiproliferative effects of kefir extracts were observed in the HMECs, while the yogurt extracts exerted antiproliferative effects on HMECs at the 5% and 10% doses. Unfermented milk extracts stimulated proliferation of MCF-7 cells and HMECs at concentrations above 0.31%. Peptide content and capillary electrophoresis analyses showed that kefir-mediated milk fermentation led to an increase in peptide concentrations and a change in peptide profiles relative to milk or yogurt. The present findings suggest that kefir extracts contain constituents that specifically inhibit the growth of human breast cancer cells, which might eventually be useful in the prevention or treatment of breast cancer.

  11. Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells

    SciTech Connect

    Yaswen, P.; Smoll, A.; Stampfer, M.R. ); Peehl, D.M. ); Trask, D.K.; Sager, R. )

    1990-10-01

    A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4 kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo({alpha})pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type {beta} increased its relative abundance. The protein encoded by NB-1 may have Ca{sup 2{sup plus}} binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined.

  12. TRIM24 Links Glucose Metabolism with Transformation of Human Mammary Epithelial Cells

    PubMed Central

    Pathiraja, Thushangi N.; Thakkar, Kaushik N.; Jiang, Shiming; Stratton, Sabrina; Liu, Zhaoliang; Gagea, Mihai; Xi, Shi; Shah, Parantu K.; Phan, Liem; Lee, Mong-Hong; Andersen, Jannik; Stampfer, Martha; Barton, Michelle C.

    2015-01-01

    Tripartite motif 24 protein (TRIM24) is a plant homeodomain (PHD)/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 cancers. PMID:25065590

  13. Of Microenvironments and Mammary Stem Cells

    SciTech Connect

    LaBarge, Mark A; Petersen, Ole W; Bissell, Mina J

    2007-06-01

    In most adult tissues there reside pools of stem and progenitor cells inside specialized microenvironments referred to as niches. The niche protects the stem cells from inappropriate expansion and directs their critical functions. Thus guided, stem cells are able to maintain tissue homeostasis throughout the ebb and flow of metabolic and physical demands encountered over a lifetime. Indeed, a pool of stem cells maintains mammary gland structure throughout development, and responds to the physiological demands associated with pregnancy. This review discusses how stem cells were identified in both human and mouse mammary glands; each requiring different techniques that were determined by differing biological needs and ethical constraints. These studies together create a robust portrait of mammary gland biology and identify the location of the stem cell niche, elucidate a developmental hierarchy, and suggest how the niche might be manipulated for therapeutic benefit.

  14. FACS Sorting Mammary Stem Cells.

    PubMed

    Iriondo, Oihana; Rábano, Miriam; Vivanco, María D M

    2015-01-01

    Fluorescent-activated cell sorting (FACS) represents one of the key techniques that have been used to isolate and characterize stem cells, including cells from the mammary gland. A combination of approaches, including recognition of cell surface antigens and different cellular activities, has facilitated the identification of stem cells from the healthy mammary gland and from breast tumors. In this chapter we describe the protocol to use FACS to separate breast cancer stem cells, but most of the general principles discussed could be applied to sort other types of cells.

  15. BRCA1/FANCD2/BRG1-Driven DNA Repair Stabilizes the Differentiation State of Human Mammary Epithelial Cells.

    PubMed

    Wang, Hua; Bierie, Brian; Li, Andrew G; Pathania, Shailja; Toomire, Kimberly; Dimitrov, Stoil D; Liu, Ben; Gelman, Rebecca; Giobbie-Hurder, Anita; Feunteun, Jean; Polyak, Kornelia; Livingston, David M

    2016-07-21

    An abnormal differentiation state is common in BRCA1-deficient mammary epithelial cells, but the underlying mechanism is unclear. Here, we report a convergence between DNA repair and normal, cultured human mammary epithelial (HME) cell differentiation. Surprisingly, depleting BRCA1 or FANCD2 (Fanconi anemia [FA] proteins) or BRG1, a mSWI/SNF subunit, caused HME cells to undergo spontaneous epithelial-to-mesenchymal transition (EMT) and aberrant differentiation. This also occurred when wild-type HMEs were exposed to chemicals that generate DNA interstrand crosslinks (repaired by FA proteins), but not in response to double-strand breaks. Suppressed expression of ΔNP63 also occurred in each of these settings, an effect that links DNA damage to the aberrant differentiation outcome. Taken together with somatic breast cancer genome data, these results point to a breakdown in a BRCA/FA-mSWI/SNF-ΔNP63-mediated DNA repair and differentiation maintenance process in mammary epithelial cells that may contribute to sporadic breast cancer development.

  16. Serotonin regulates β-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

    PubMed

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

    2015-01-01

    We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses β-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of β-casein protein expression. β-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased β-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. β-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, β-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates β-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.

  17. Distribution of obestatin and ghrelin in human tissues: immunoreactive cells in the gastrointestinal tract, pancreas, and mammary glands.

    PubMed

    Grönberg, Malin; Tsolakis, Apostolos V; Magnusson, Linda; Janson, Eva T; Saras, Jan

    2008-09-01

    Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immunoreactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.

  18. Sclerotium rolfsii lectin induces stronger inhibition of proliferation in human breast cancer cells than normal human mammary epithelial cells by induction of cell apoptosis.

    PubMed

    Savanur, Mohammed Azharuddin; Eligar, Sachin M; Pujari, Radha; Chen, Chen; Mahajan, Pravin; Borges, Anita; Shastry, Padma; Ingle, Arvind; Kalraiya, Rajiv D; Swamy, Bale M; Rhodes, Jonathan M; Yu, Lu-Gang; Inamdar, Shashikala R

    2014-01-01

    Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.

  19. Bovine mammary stem cells: new perspective for dairy science.

    PubMed

    Martignani, E; Cravero, D; Miretti, S; Accornero, P; Baratta, M

    2014-01-01

    Mammary stem cells provide opportunities for the cyclic remodelling of the bovine mammary gland. Therefore, understanding the character and regulation of mammary stem cells is important for increasing animal health and productivity. The exciting possibility that stem cell expansion can influence milk production is currently being investigated by several researchers. In fact, appropriate regulation of mammary stem cells could hopefully benefit milk yield, persistency of lactation, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and regulate the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies, which represents a limitation for the management of animal welfare. Interestingly, different studies recently reported the identification of putative mammary stem cells in human breast milk. The possible identification of primitive cell types within cow's milk may provide a non-invasive source of relevant mammary cells for a wide range of applications. In this review, we have summarized the main achievements in this field for dairy cow science and described the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period by regulating mammary stem cells.

  20. 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.

  1. The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay

    PubMed Central

    Weaver, V.M.; Fischer, A.H.; Peterson, O.W.; Bissell, M.J.

    2010-01-01

    The extracellular matrix (ECM) is a dominant regulator of tissue development and homeostasis. “Designer microenvironments” in culture and in vivo model systems have shown that the ECM regulates growth, differentiation, and apoptosis in murine and human mammary epithelial cells (MEC) through a hierarchy of transcriptional events involving the intricate interplay between soluble and physical signaling pathways. Furthermore, these studies have shown that these pathways direct and in turn are influenced by the tissue structure. Tissue structure is directed by the cooperative interactions of the cell–cell and cell–ECM pathways and can be modified by stromal factors. Not surprisingly then, loss of tissue structure and alterations in ECM components are associated with the appearance and dissemination of breast tumors, and malignancy is associated with perturbations in cell adhesion, changes in adhesion molecules, and a stromal reaction. Several lines of evidence now support the contention that the pathogenesis of breast cancer is determined (at least in part) by the dynamic interplay between the ductal epithelial cells, the microenvironment, and the tissue structure (acini). Thus, to understand the mechanisms involved in carcinogenesis, the role of the microenvironment (ECM as well as the stromal cells) with respect to tissue structure should be considered and studied. Towards this goal, we have established a unique human MEC model of tumorigenesis, which in concert with a three-dimensional assay, recapitulates many of the genetic and morphological changes observed in breast cancer in vivo. We are currently using this system to understand the role of the microenvironment and tissue structure in breast cancer progression. PMID:9164652

  2. Morphological and histological characteristics of mammary dysplasias occurring in cell dissociation-derived murine mammary outgrowths

    SciTech Connect

    Ethier, S.P.; Adams, L.M.; Ullrich, R.L.

    1984-10-01

    The morphological and histological characteristics of ductal dysplasias that were observed in mammary outgrowths derived from monodispersed mammary cells of carcinogen-treated mice are described. Mammary outgrowths were derived by injecting either 10(4) or 10(5) enzymatically dissociated mammary cells, obtained from control or carcinogen-treated BALB/c mice, into gland-free mammary fat pads of syngeneic hosts. The mammary dysplasias observed varied considerably in morphological and histological characteristics. The majority of the lesions were ductal in origin and were associated with epithelial hyperplasia which ranged from mild hyperplasia, in which only a few extra layers of epithelium were present, to severe hyperplasia, in which the ducts and end buds were occluded and distended with epithelial cells. In addition, papillary and lobular lesions were observed which were also associated with varying degrees of hyperplasia. The range of mammary dysplasias observed in these outgrowths closely resembles that of lesions associated with the pathogenesis of mammary carcinoma in mice, rats, and humans.

  3. KISS1R induces invasiveness of estrogen receptor-negative human mammary epithelial and breast cancer cells.

    PubMed

    Cvetkovic, Donna; Dragan, Magdalena; Leith, Sean J; Mir, Zuhaib M; Leong, Hon S; Pampillo, Macarena; Lewis, John D; Babwah, Andy V; Bhattacharya, Moshmi

    2013-06-01

    Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor α (ERα)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ERα-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ERα-positive MCF7 and T47D breast cancer cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ERα in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis.

  4. 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

  5. Role of P13 Kinase Signaling Pathways in Polarity Determination of Human Mammary Epithelial Cells Grown in Three-Dimensional Extracellular Matrix

    DTIC Science & Technology

    2005-09-01

    morphogenesis, wound healing , and immune- cell trafficking (Friedl and Brocker 2000; Friedl and Wolf 2003). Unlike physiological processes of cell...Boyden chambers (Figure 1D, n=8). Since altered cell motility is the driving force of invasiveness, wound - healing and cell migration assays c were...AD Award Number: DAMD17-03-1-0099 TITLE: Role of P13 Kinase Signaling Pathways in Polarity Determination of Human Mammary Epithelial Cells Grown in

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

    SciTech Connect

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

    2009-06-03

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

  7. Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

    PubMed

    Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

    2015-02-01

    During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer.

  8. Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors.

    PubMed

    Nishi, M; Sakai, Y; Akutsu, H; Nagashima, Y; Quinn, G; Masui, S; Kimura, H; Perrem, K; Umezawa, A; Yamamoto, N; Lee, S W; Ryo, A

    2014-01-30

    Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44(+)/CD24(low) cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

  9. Retinoids modulate expression of the endocytic partners megalin, cubilin, and disabled-2 and uptake of vitamin D-binding protein in human mammary cells.

    PubMed

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

    2008-07-01

    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.

  10. Chromatin Regulation of the EGFR Locus in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2006-05-01

    milk proteins, including α-casein, γ-casein, whey acidic protein (WAP), and β-lactoglobulin have been shown to be regulated by ECM and lactogenic...these two milk proteingenes. Thus, ECM-and prolactin-regulated transcription of the mammary-specific case in genes requires the concertedaction of...two milk protein genes. Thus, ECM-regulated transcription of the mammary-specific casein genes requires the concerted action of histone acetylation

  11. 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

  12. 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.

  13. Different Mutant/Wild-Type p53 Combinations Cause a Spectrum of Increased Invasive Potential in Nonmalignant Immortalized Human Mammary Epithelial Cells1

    PubMed Central

    Junk, Damian J; Vrba, Lukas; Watts, George S; Oshiro, Marc M; Martinez, Jesse D; Futscher, Bernard W

    2008-01-01

    Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease. PMID:18472962

  14. Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology.

    PubMed

    Singh, Dipika; Pannier, Angela K; Zempleni, Janos

    2011-06-01

    Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.

  15. Similarity of GATA-3 Expression between Rat and Human Mammary Glands.

    PubMed

    Kinoshita, Yuichi; Yoshizawa, Katsuhiko; Emoto, Yuko; Yuki, Michiko; Yuri, Takashi; Shikata, Nobuaki; Tsubura, Airo

    2014-07-01

    The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. In particular, GATA-3 is necessary for mammary gland maturation and is a useful marker in the characterization of mammary carcinoma in humans. The expression of GATA-3 protein in normal mammary glands, fibroadenomas and carcinomas was immunohistochemically compared in female rats and humans. In normal mammary glands of rats and humans, scattered luminal cells in the acini and whole ductal epithelial cells were positive for GATA-3 in the nuclei. No positive cells were detected in rat or human fibroadenomas. In rat and human mammary carcinomas, the nuclei of proliferating luminal-derived cancer cells expressed GATA-3. Therefore, GATA-3 protein is a candidate marker for mammary carcinoma in rats as well as humans.

  16. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    PubMed

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  17. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer.

    PubMed

    Spike, Benjamin T; Engle, Dannielle D; Lin, Jennifer C; Cheung, Samantha K; La, Justin; Wahl, Geoffrey M

    2012-02-03

    Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate, and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and coexpress multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms, including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis, and therapy.

  18. Generation of breast cancer stem cells by steroid hormones in irradiated human mammary cell lines.

    PubMed

    Vares, Guillaume; Cui, Xing; Wang, Bing; Nakajima, Tetsuo; Nenoi, Mitsuru

    2013-01-01

    Exposure to ionizing radiation was shown to result in an increased risk of breast cancer. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs). In order to assess whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs, we measured by flow cytometry the proportion of CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR)-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells, progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the downregulation of miR-22 and miR-29c expression), which resulted in increased proportions of radiation-resistant tumor-initiating CSCs.

  19. Primary Human Mammary Epithelial Cells Endocytose HIV-1 and Facilitate Viral Infection of CD4+ T Lymphocytes ▿

    PubMed Central

    Dorosko, Stephanie M.; Connor, Ruth I.

    2010-01-01

    The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4+ cells throughout the breast-feeding period, it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4+ cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4+ target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-, CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4+ target cells and to activate resting CD4+ T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4, CCR5, CXCR4, and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found, HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4+ T cells. In addition, MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall, our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4+ target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4+ T lymphocytes in vivo. PMID:20702626

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

    PubMed Central

    Girard, Brian J.; Regan Anderson, Tarah M.; Welch, Siya Lem; Nicely, Julie; Seewaldt, Victoria L.; Ostrander, Julie H.

    2015-01-01

    Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness. PMID:25789479

  1. Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice.

    PubMed

    Wang, Qing; Hao, Siguo; Ma, Liyuan; Zhang, Wenhao; Wan, Jiangbo; Deng, Xiaohui

    2015-10-01

    Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland 'bioreactor' is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P < 0.05). While hFVIIIBD was present in multiple organs in CMV-hFVIIIBD mice, P1A3-hFVIIIBD mice showed negligible hFVIIIBD expression in organs other than the mammary glands. This study demonstrated that the mammary gland-specific P1A3-hFVIIIBD vector was more suitable for the generation of hFVIIIBD mammary gland bioreactor.

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

    PubMed

    van Meeuwen, J A; Korthagen, N; de Jong, P C; Piersma, A H; van den Berg, M

    2007-06-15

    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 developed 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(50)s decreased in the following order: estadiol (4*10(-3) nM)>biochanin A (9 nM)>genistein (32 nM)>testosterone (46 nM)>naringenin (287 nM)>apigenin (440 nM)>chrysin (4 microM). The potency to inhibit aromatase derived from their IC(50)s decreased in the following order: chrysin (1.5 microM)>naringenin (2.2 microM)>genistein (3.6 microM)>apigenin (4.1 microM)>biochanin A (25 microM)>quercetin (30 microM). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1-10 microM). Results from co-cultures did not elucidate the dominant effect of these compounds. MCF-7 cell proliferation occurs at concentrations that are not uncommon in blood of individuals using food

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

    SciTech Connect

    Meeuwen, J.A. van . E-mail: J.A.vanMeeuwen@iras.uu.nl; Korthagen, N.; Jong, P.C. de; Piersma, A.H.; Berg, M. van den

    2007-06-15

    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 developed 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 in blood

  4. Mammary Stem Cell Research in Veterinary Science: An Update

    PubMed Central

    Borena, Bizunesh M.; Bussche, Leen; Burvenich, Christian; Duchateau, Luc

    2013-01-01

    The mammary gland is an organ with a remarkable regenerative capacity that can undergo multiple cycles of proliferation, lactation, and involution. Growing evidence suggests that these changes are driven by the coordinated division and differentiation of mammary stem cell populations (MaSC). Whereas information regarding MaSC and their role in comparative mammary gland physiology is readily available in human and mice, such information remains scarce in most veterinary mammal species such as cows, horses, sheep, goats, pigs, and dogs. We believe that a better knowledge on the MaSC in these species will not only help to gain more insights into mammary gland (patho) physiology in veterinary medicine, but will also be of value for human medicine. Therefore, this review summarizes the current knowledge on stem cell isolation and characterization in different mammals of veterinary importance. PMID:23360296

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Role of p53 Mammary Epithelial Cell Senescence

    DTIC Science & Technology

    2005-05-01

    susceptibility to oncogenesis exist in the normal mammary tissue, the milk -forming ducts of the mammary gland (for review [2]). Coupled with information...HMECs [25]. proportion of preselection HMECs [8]. The senescence associated with the ’selection’ phase in Human milk is an easily available source of...which has precluded their detailed biochemical proliferation at this stage is dependent on the pRb/p16 study [2,18]. Most of the work on milk cells has

  7. A Comprehensive, Multi-Scale Dynamical Model of ErbB Receptor Signal Transduction in Human Mammary Epithelial Cells

    PubMed Central

    Helikar, Tomáš; Kochi, Naomi; Kowal, Bryan; Dimri, Manjari; Naramura, Mayumi; Raja, Srikumar M.; Band, Vimla; Band, Hamid; Rogers, Jim A.

    2013-01-01

    The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF). Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis. PMID:23637902

  8. Human Mammary Epithelial Cell Transformation by Rho GTPase Through a Novel Mechanism

    DTIC Science & Technology

    2009-08-01

    and scanning were performed by Microarray Core Facility, Northwestern University. Affymetrix Human Genome U133 Plus 2.0 chips (containing >47,000...annotation of the HG- U133 Plus 2 microarray was updated using the Entrez gene database at the National Center for Biotechnology Information (NCBI). Raw...gene expression profiles of normal hMECs with those of cells immortalized using RhoA-WT, G14V, or T37A using the Affymetrix Human Genome U133 Plus 2.0

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

    SciTech Connect

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

    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 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. 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.

  10. 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.

  11. Stem cells and the developing mammary gland.

    PubMed

    Makarem, Maisam; Spike, Benjamin T; Dravis, Christopher; Kannan, Nagarajan; Wahl, Geoffrey M; Eaves, Connie J

    2013-06-01

    The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.

  12. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

    PubMed

    Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen

    2007-09-15

    Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  13. Milk fat conjugated linoleic acid (CLA) inhibits growth of human mammary MCF-7 cancer cells.

    PubMed

    O'Shea, M; Devery, R; Lawless, F; Murphy, J; Stanton, C

    The relationship between growth and the antioxidant enzyme defence system in human MCF-7 (breast) cancer cells treated with bovine milk fat enriched with conjugated linoleic acid (CLA) was studied. Milk enriched in CLA was obtained from cows on pasture supplemented with full fat rapeseeds and full fat soyabeans (1). Cell number decreased up to 90% (p < 0.05) and lipid peroxidation increased 15-fold (p < 0.05) following incubation of MCF-7 cells for 8 days with increasing levels of milk fat yielding CLA concentrations between 16.9 and 22.6 ppm. Growth suppression and prooxidant effects of milk fat CLA were independent of the variable composition of the milk fat samples, suggesting that CLA was the active ingredient in milk fat responsible for the cytotoxic effect. Mixtures containing isomers of CLA (c9, t11-, t10, c12-, c11, t13- and minor amounts of other isomers) and linoleic acid (LA) at similar concentrations to the milk fat samples were as effective at inhibiting growth and stimulating peroxidation of MCF-7 cells as the milk fatty acids. Incubation of the cells with the c9, t11 CLA isomer (20 ppm) or the mixture of CLA isomers (20 ppm) for 8 days resulted in a 60% decrease (p < 0.05) in viability compared with untreated controls and was significantly (p < 0.05) more effective than incubation with the t10, c12 CLA isomer (20 ppm), which caused only a 15% decrease in cell numbers under similar conditions. A 25% increase (p < 0.05) in cell proliferation occurred when LA (20 ppm) alone was incubated with MCF-7 cells for 8 days. 14C-CLA was preferentially incorporated into the phospholipid fraction of the MCF-7 cell lipids in a dose-dependent manner and CLA accumulated in cell membranes more efficiently when the cells were incubated in the presence of milk fat than the c9, t11 synthetic CLA isomer. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities were induced in MCF-7 cells exposed to milk fat (containing 16.9-22.6 ppm CLA) over 8

  14. Immunohistochemical characterization of mammary squamous cell carcinoma of the dog.

    PubMed

    Sassi, Francesco; Sarli, Giuseppe; Brunetti, Barbara; Morandi, Federico; Benazzi, Cinzia

    2008-11-01

    Squamous cell carcinoma of the mammary gland is rare in both veterinary and human medicine. Whereas human metaplastic and squamous variants are known, the objectives of the current study were to ascertain the presence of such entities in canine mammary tumors and to distinguish them from other (epidermal, sweat gland) squamous tumors that may develop in the same area. A panel of antibodies (anti-cytokeratin [CK] 19, CK 14, CK 5/6, pancytokeratin, and vimentin) was used on 18 mammary gland malignancies with squamous features and 16 malignant skin tumors (11 squamous cell carcinomas of the skin and 5 sweat glands). Fifteen of the 18 mammary carcinomas were classified as metaplastic carcinomas, and the remaining 3 were classified as squamous cell carcinomas. The 2 most useful markers to establish the histogenesis of mammary tumors were pancytokeratin and CK 19. All other antibodies were equally expressed (CK 14 and 5/6) in all histotypes. The antibody panel discriminated primary epidermal squamous tumors (pancytokeratin positive and CK 19 negative) from gland-derived squamous neoplasms (pancytokeratin positive and CK 19 positive) but failed to distinguish primary mammary tumors from other squamous tumors of glandular origin.

  15. A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function.

    PubMed

    Wang, Xiuli; Sun, Lin; Maffini, Maricel V; Soto, Ana; Sonnenschein, Carlos; Kaplan, David L

    2010-05-01

    Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel/collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein alpha- and -beta mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.

  16. Analysis of Immune Cells from Human Mammary Ductal Epithelial Organoids Reveals Vδ2+ T Cells That Efficiently Target Breast Carcinoma Cells in the Presence of Bisphosphonate.

    PubMed

    Zumwalde, Nicholas A; Haag, Jill D; Sharma, Deepak; Mirrielees, Jennifer A; Wilke, Lee G; Gould, Michael N; Gumperz, Jenny E

    2016-04-01

    Developing strategies to enhance cancer prevention is a paramount goal, particularly given recent concerns about surgical treatment of preinvasive states such as ductal carcinoma in situ. Promoting effective immunosurveillance by leukocytes that scan for nascent neoplastic transformations represents a potential means to achieve this goal. Because most breast cancers arise within the ductal epithelium, enhancing protective immunosurveillance will likely necessitate targeting one or more of the distinctive lymphocyte types found in these sites under normal conditions. Here, we have characterized the intraepithelial lymphocyte compartment of non-cancerous human breast tissue and identified a subset of T lymphocytes that can be pharmacologically targeted to enhance their responses to breast cancer cells. Specifically, Vδ2(+) γδ T cells were consistently present in preparations of mammary ductal epithelial organoids and they proliferated in response to zoledronic acid, an aminobisphosphonate drug. Vδ2(+) T cells from breast ductal organoids produced the antitumor cytokine IFNγ and efficiently killed bisphosphonate-pulsed breast carcinoma cells. These findings demonstrate the potential for exploiting the ability of Vδ2(+) γδ T cells to respond to FDA-approved bisphosphonate drugs as a novel immunotherapeutic approach to inhibit the outgrowth of breast cancers.

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

    PubMed

    Mohammad, Mahmoud A; Haymond, Morey W

    2013-09-15

    Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal 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 secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-γ decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.

  18. 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

  19. Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells.

    PubMed

    Nakatani, Hajime; Aoki, Naohito; Nadano, Daita; Matsuda, Tsukasa

    2011-01-01

    The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.

  20. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract

    SciTech Connect

    Hammond, S.L.; Ham, R.G.; Stampfer, M.R.

    1984-09-01

    A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, (MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E/sub 1/ and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three of four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelia morphology, distribution of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells. 31 references, 3 figures, 4 tables.

  1. 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

    Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a...17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c. THIS PAGE U UU 14 19b...timed pregnant female mice, obtain embryos from El8.5, dissect mammary rudiments, isolate fMaSC and fStroma by methods they have developed. The cells

  2. Comparison of several radiation effects in human MCF10A mammary epithelial cells cultured as 2D monolayers or 3D acinar stuctures in matrigel.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Peng, Yuanlin; Chuang, Eric Y; Bedford, Joel S

    2009-06-01

    It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of gamma-H2AX foci in cells after single acute gamma-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.

  3. Activation of Alternative Wnt Signaling Pathways in Human Mammary Gland and Breast Cancer Cells

    DTIC Science & Technology

    2006-06-01

    signalling, the antagonistic role of Wnt5a on canonical Wnt signalling, and the fact that the genes regulated by either of these pathways differ in...differentiation, apoptosis, and migration. Wnt/Frizzled signaling is now linked to human hereditary disorders with retinal vascular defects, implicating...www.physiologyonline.org fact that the role of FrzA in vascular biology is not well understood. Wnt Signaling Comes into Play in Human Vascular

  4. Basis of Persistent Microenvironment Perturbation in Irradiated Human Mammary Epithelial Cells

    DTIC Science & Technology

    2005-07-01

    SUBJECT TERMS Genomic instability, Ionizing radiation , Cellular structure, Growth factors/Cytokines 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18...exploratory studies will define non-mutational mechanisms by which ionizing radiation , a known carcinogen of human breast, affects carcinogenesis... ionizing radiation represents a well- established carcinogen. Epidemiologic data demonstrates that there is a significantly increased risk of breast

  5. Hormone signaling requirements for the conversion of non-mammary mouse cells to mammary cell fate(s) in vivo.

    PubMed

    Boulanger, Corinne A; Rosenfield, Sonia M; George, Andrea L; Smith, Gilbert H

    2015-06-01

    Mammotropic hormones and growth factors play a very important role in mammary growth and differentiation. Here, hormones including Estrogen, Progesterone, Prolactin, their cognate receptors, and the growth factor Amphiregulin, are tested with respect to their roles in signaling non-mammary cells from the mouse to redirect to mammary epithelial cell fate(s). This was done in the context of glandular regeneration in pubertal athymic female mice. Our previous studies demonstrated that mammary stem cell niches are recapitulated during gland regeneration in vivo. During this process, cells of exogenous origin cooperate with mammary epithelial cells to form mammary stem cell niches and thus respond to normal developmental signals. In all cases tested with the possible exception of estrogen receptor alpha (ER-α), hormone signaling is dispensable for non-mammary cells to undertake mammary epithelial cell fate(s), proliferate, and contribute progeny to chimeric mammary outgrowths. Importantly, redirected non-mammary cell progeny, regardless of their source, have the ability to self-renew and contribute offspring to secondary mammary outgrowths derived from transplanted chimeric mammary fragments; thus suggesting that some of these cells are capable of mammary stem cell/progenitor functions.

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

    DTIC Science & Technology

    1990-07-13

    cycle regulation of histone mRNAs ( Luscher and SchumperU, 1987). A 180 nucleotide fragment derived from the 3’ 19 end of exon 1 of c-myc reduces...Oncol. 4: 1321-1325. Luscher , B., and D. SchumperU. (1987) RNA 3’ Processing Regulates Histone mRNA Levels in a Mammalian Cell Cycle Mutant. A...P.W.J., M. Dieckmann, C Rhodes, and R Berg . (1977) Labeling Deoxyribonucleic Add to High Spedfic Activity in Vitro by Nick Translation witii DNA

  7. Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors

    PubMed Central

    Callahan, Robert; Mudunuri, Uma; Bargo, Sharon; Raafat, Ahmed; McCurdy, David; Boulanger, Corinne; Lowther, William; Stephens, Robert; Luke, Brian T.; Stewart, Claudia; Wu, Xiaolin; Munroe, David; Smith, Gilbert H.

    2012-01-01

    The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  9. Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics.

    PubMed

    Choudhury, Sibgat; Almendro, Vanessa; Merino, Vanessa F; Wu, Zhenhua; Maruyama, Reo; Su, Ying; Martins, Filipe C; Fackler, Mary Jo; Bessarabova, Marina; Kowalczyk, Adam; Conway, Thomas; Beresford-Smith, Bryan; Macintyre, Geoff; Cheng, Yu-Kang; Lopez-Bujanda, Zoila; Kaspi, Antony; Hu, Rong; Robens, Judith; Nikolskaya, Tatiana; Haakensen, Vilde D; Schnitt, Stuart J; Argani, Pedram; Ethington, Gabrielle; Panos, Laura; Grant, Michael; Clark, Jason; Herlihy, William; Lin, S Joyce; Chew, Grace; Thompson, Erik W; Greene-Colozzi, April; Richardson, Andrea L; Rosson, Gedge D; Pike, Malcolm; Garber, Judy E; Nikolsky, Yuri; Blum, Joanne L; Au, Alfred; Hwang, E Shelley; Tamimi, Rulla M; Michor, Franziska; Haviv, Izhak; Liu, X Shirley; Sukumar, Saraswati; Polyak, Kornelia

    2013-07-03

    Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44(+) progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44(+)p27(+) cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27(+) cells and their proliferation. Our results suggest that pathways controlling p27(+) mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

  10. Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

    SciTech Connect

    Wang Enxiu; Obeng-Adjei, Nyamekye; Ying Qihua; Davey, Robert A.; Ross, Susan R.

    2008-11-25

    Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment.

  11. Distinct FAK activities determine progenitor and mammary stem cell characteristics

    PubMed Central

    Luo, Ming; Zhao, Xiaofeng; Chen, Song; Liu, Suling; Wicha, Max S.; Guan, Jun-Lin

    2013-01-01

    Mammary stem (MaSCs) and progenitor cells are important for mammary gland development and maintenance and may give rise to mammary cancer stem cells (MaCSCs). Yet there remains limited understanding of how these cells contribute to tumorigenesis. Here we show that conditional deletion of focal adhesion kinase (FAK) in embryonic mammary epithelial cells (MaECs) decreases luminal progenitors (LPs) and basal MaSCs, reducing their colony-forming and regenerative potentials in a cell autonomous manner. Loss of FAK kinase activity in MaECs specifically impaired LP proliferation and alveologenesis, whereas a kinase-independent activity of FAK supported ductal invasion and basal MaSC activity. Deficiency in LPs suppressed tumorigenesis and MaCSC formation in a mouse model of breast cancer. In contrast to the general inhibitory effect of FAK attenuation, inhibitors of FAK kinase preferentially inhibited proliferation and tumorsphere formation of LP-like, but not MaSC-like, human breast cancer cells. Our findings establish distinct kinase dependent and independent activities of FAK that differentially regulate LPs and basal MaSCs. We suggest that targeting these distinct functions may tailor therapeutic strategies to address breast cancer heterogeneity more effectively. PMID:23832665

  12. bcl-2 overexpression inhibits cell death and promotes the morphogenesis, but not tumorigenesis of human mammary epithelial cells [published erratum appears in J Cell Biol 1995 Nov;131(4):following 1121

    PubMed Central

    1995-01-01

    Overexpression of the B cell leukemia/lymphoma-2 (bcl-2) gene has been shown to confer a survival advantage on cells by inhibiting apoptosis. In epithelia, the bcl-2 gene is also related to development and differentiation, and the protein is strongly expressed in the embryo in the epithelial cells of the developing mammary gland. To investigate directly the effect of bcl-2 on human epithelial cells, we used an amphotropic recombinant retrovirus to introduce the gene into nontumorigenic cell lines developed from luminal epithelial cells cultured from milk. Here we demonstrate that while bcl-2 overexpression does not directly induce the tumorigenic phenotype, it provides a survival advantage to the mammary epithelial cells by inhibiting cell death at confluence or under conditions of serum starvation, bcl-2 can also affect the phenotype of the original epithelial cells, and promote epithelial-mesenchymal conversion, accompanied by loss of the cell adhesion molecules E-cadherin and alpha 2 beta 1 integrin. The extent of the epithelial-mesenchymal conversion varies with small differences in the phenotype of the parental line and with the level of expression of Bcl-2 and in some cases cell lines emerge with a mixed phenotype. The increased survival of Bcl-2-expressing cells at confluence results in multilayering, and the development of three- dimensional structures. Where a mixed phenotype is observed these structures consist of an outer layer of polarized epithelial cells separated by a basement membrane-like layer from an inner mass of fibroblastoid cells. Branching morphogenesis of bcl-2 transfectants is also observed in collagen gels (in the absence of fibroblast growth factors). The results strongly indicate that by increasing their survival under restrictive growth conditions, and by modifying the epithelial phenotype, bcl-2 can influence the specific morphogenetic behavior of mammary epithelial cells. PMID:7775580

  13. 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

  14. 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.

  15. Expression of different phenotypes in cell lines from canine mammary spindle-cell tumours and osteosarcomas indicating a pluripotent mammary stem cell origin.

    PubMed

    Hellmén, E; Moller, M; Blankenstein, M A; Andersson, L; Westermark, B

    2000-06-01

    Mammary spindle-cell tumours and sarcomas seem to be restricted to dogs and humans. Two cell lines from spontaneous primary canine mammary spindle-cell tumours (CMT-U304 and CMT-U309) and two cell lines from spontaneous primary canine mammary osteosarcomas (CMT-U334 and CMT-U335) were established to study the mesenchymal phenotypes of mammary tumours in the female dog. The cells from the spindle-cell tumours expressed cytokeratin, vimentin and smooth muscle actin filaments. When these cells were inoculated subcutaneously into female and male nude mice they formed different types of mesenchymal tumours such as spindle-cell tumours, fibroma and rhabdomyoid tumours (n = 6/8). The cells from the osteosarcomas expressed vimentin filaments and also formed different types of mesenchymal tumours such as chondroid, rhabdomyoid, smooth muscle-like and spindle-cell tumours (n = 6/10). The cell lines CMT-U304, CMT-U309 and CMT-U335 had receptors for progesterone but none of the four cell lines had receptors for estrogen. All four cell lines and their corresponding primary tumours showed identical allelic patterns in microsatellite analysis. By in situ hybridization with genomic DNA we could verify that all formed tumours but one were of canine origin. Our results support the hypothesis that canine mammary tumours are derived from pluripotent stem cells.

  16. Human breast adipose‑derived stem cells: characterization and differentiation into mammary gland‑like epithelial cells promoted by autologous activated platelet‑rich plasma.

    PubMed

    Cui, Shi-En; Li, Hong-Mian; Liu, Da-Lie; Nan, Hua; Xu, Kun-Ming; Zhao, Pei-Ran; Liang, Shuang-Wu

    2014-08-01

    Human adipose‑derived stem cells (ASCs) isolated from various body sites have been widely investigated in basic and clinical studies. However, ASCs derived from human breast tissue (hbASCs) have not been extensively investigated. In order to expand our understanding of hbASCs and examine their potential applications in stem cell research and cell‑based therapy, hbASCs were isolated from discarded surgical fat tissue following reduction mammoplasty and a comprehensive characterization of these hbASCs was performed, including analysis of their cellular morphology, growth features, cell surface protein markers and multilineage differentiation capacity. These hbASCs expressed cluster of differentiation (CD)44, CD49d, CD90 and CD105, but did not express CD31 and CD34. Subsequently, the hbASCs were differentiated into adipocytes, osteocytes and chondrocytes in vitro. In order to examine the potential applications of hbASCs in breast reconstruction, an approach to promote in vitro differentiation of hbASCs into mammary gland‑like epithelial cells (MGECs) was developed using activated autologous platelet‑rich plasma (PRP). A proliferation phase and a subsequent morphological conversion phase were observed during this differentiation process. PRP significantly promoted the growth of hbASCs in the proliferation phase and increased the eventual conversion rate of hbASCs into MGECs. Thus, to the best of our knowledge, the present study provided the first comprehensive characterization of hbASCs and validated their multipotency. Furthermore, it was revealed that activated autologous PRP was able to enhance the differentiation efficiency of hbASCs into MGECs. The present study and other studies of hbASCs may aid the development of improved breast reconstruction strategies.

  17. Canine mammary tumour cell lines established in vitro.

    PubMed

    Hellmén, E

    1993-01-01

    Mammary tumours are the most common tumours in the female dog. The tumours have a complex histology and exist in epithelial, mixed and mesenchymal forms. To study the biology of canine mammary tumours, five cell lines have been established and characterized. The results indicate that canine mammary tumours might be derived from mammary stem cells and that the tumour growth is independent of oestrogens. The established canine mammary tumour cell lines will be valuable tools in further studies of the histogenesis and pathogenesis of these tumours.

  18. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  19. Metabotropic glutamate receptor 1 disrupts mammary acinar architecture and initiates malignant transformation of mammary epithelial cells

    PubMed Central

    Teh, Jessica L. F.; Shah, Raj; La Cava, Stephanie; Dolfi, Sonia C.; Mehta, Madhura S.; Kongara, Sameera; Price, Sandy; Ganesan, Shridar; Reuhl, Kenneth R.; Hirshfield, Kim M.

    2016-01-01

    Metabotropic glutamate receptor 1 (mGluR1/Grm1) is a member of the G-protein-coupled receptor superfamily, which was once thought to only participate in synaptic transmission and neuronal excitability, but has more recently been implicated in non-neuronal tissue functions. We previously described the oncogenic properties of Grm1 in cultured melanocytes in vitro and in spontaneous melanoma development with 100 % penetrance in vivo. Aberrant mGluR1 expression was detected in 60–80 % of human melanoma cell lines and biopsy samples. As most human cancers are of epithelial origin, we utilized immortalized mouse mammary epithelial cells (iMMECs) as a model system to study the transformative properties of Grm1. We introduced Grm1 into iMMECs and isolated several stable mGluR1-expressing clones. Phenotypic alterations in mammary acinar architecture were assessed using three-dimensional morphogenesis assays. We found that mGluR1-expressing iMMECs exhibited delayed lumen formation in association with decreased central acinar cell death, disrupted cell polarity, and a dramatic increase in the activation of the mitogen-activated protein kinase pathway. Orthotopic implantation of mGluR1-expressing iMMEC clones into mammary fat pads of immunodeficient nude mice resulted in mammary tumor formation in vivo. Persistent mGluR1 expression was required for the maintenance of the tumorigenic phenotypes in vitro and in vivo, as demonstrated by an inducible Grm1-silencing RNA system. Furthermore, mGluR1 was found be expressed in human breast cancer cell lines and breast tumor biopsies. Elevated levels of extracellular glutamate were observed in mGluR1-expressing breast cancer cell lines and concurrent treatment of MCF7 xenografts with glutamate release inhibitor, riluzole, and an AKT inhibitor led to suppression of tumor progression. Our results are likely relevant to human breast cancer, highlighting a putative role of mGluR1 in the pathophysiology of breast cancer and the potential

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

    PubMed Central

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

    2003-01-01

    Background The family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we examined its cellular localization in vivo using immunohistochemistry. Methods Extracts of whole mammary glands from wild type and Id-1 null mutant mice, and tissue sections from paraffin-embedded mouse mammary glands from various developmental stages and normal human breast were subjected to immunoblot and immunohistochemical analyses, respectively. In both these procedures, an anti-Id-1 rabbit polyclonal antibody was used for detection of Id-1. Results In immunoblot analyses, using whole mammary gland extracts, Id-1 was detected. In immunohistochemical analyses, however, Id-1 was not detected in the luminal epithelial cells of mammary glands during any stage of development, but it was detected in vascular endothelial cells. Conclusion Id-1 is not expressed in the luminal epithelial cells of mammary glands. PMID:12631395

  1. 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.

  2. Transcriptional profiles of benzo(a)pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin.

    PubMed

    John, Kaarthik; Keshava, Channa; Richardson, Diana L; Weston, Ainsley; Nath, Joginder

    2008-04-02

    Benzo(a)pyrene (BP) exposure causes alterations in gene expression in normal human mammary epithelial cells (NHMECs). This study used Affymetrix Hu-Gene133A arrays, with 14,500 genes represented, to evaluate modulation of BP-induced gene expression by chlorophyllin in six NHMEC strains derived from different donors. A major goal was to seek potential biomarkers of carcinogen exposure and how they behave in the presence of a chemopreventive agent. NHMECs (passage 6 and 70% confluence) were exposed for 24h to either vehicle control, or BP, or chlorophyllin followed by BP and chlorophyllin together. BP exposure resulted in approximately 3-fold altered expression of 49 genes in at least one of the six NHMEC strains. When cells were exposed to chlorophyllin pre-treatment followed by BP plus chlorophyllin, expression of 125 genes was similarly altered. Genes in the functional categories of xenobiotic metabolism, cell signaling, cell motility, cell proliferation, cellular transcription, metabolism, cell cycle control, apoptosis and DNA repair were identified. Only CYP1B1 and ALDH1A3 were consistently up-regulated by approximately 3-fold in most of the cell strains (at least 4) when exposed to BP. Cluster analysis identified a suite of 13 genes induced by BP where induction was mitigated in the presence of chlorophyllin. Additionally, cluster analysis identified a suite of 16 genes down-regulated by BP where induction was partially restored in the presence of chlorophyllin.

  3. 4-Hydroxyestradiol induces oxidative stress and apoptosis in human mammary epithelial cells: possible protection by NF-kappaB and ERK/MAPK.

    PubMed

    Chen, Zhi-Hua; Na, Hye-Kyung; Hurh, Yeon-Jin; Surh, Young-Joon

    2005-10-01

    Catechol estrogens, the hydroxylated metabolites of 17beta-estradiol (E2), have been considered to be implicated in estrogen-induced carcinogenesis. 4-Hydroxyestradiol (4-OHE2), an oxidized metabolite of E2 formed preferentially by cytochrome P450 1B1, reacts with DNA to form depurinating adducts thereby exerting genotoxicity and carcinogenicity. 4-OHE2 undergoes 2-electron oxidation to quinone via semiquinone, and during this process, reactive oxygen species (ROS) can be generated to cause DNA damage and cell death. In the present study, 4-OHE2 was found to elicit cytotoxicity in cultured human mammary epithelial (MCF-10A) cells, which was blocked by the antioxidant trolox. MCF-10A cells treated with 4-OHE2 exhibited increased intracellular ROS accumulation and 8-oxo-7,8-dihydroxy-2'-deoxyguanosine formation, and underwent apoptosis as determined by poly(ADP-ribose)polymerase cleavage and disruption of mitochondrial transmembrane potential. The redox-sensitive transcription factor nuclear factor kappaB (NF-kappaB) was transiently activated by 4-OHE2 treatment. Cotreatment of MCF-10A cells with the NF-kappaB inhibitor, L-1-tosylamido-2-phenylethyl chloromethyl ketone, exacerbated 4-OHE2-induced cell death. 4-OHE2 also caused transient activation of extracellular signal-regulated protein kinases (ERK) involved in transmitting cell survival or death signals. A pharmacological inhibitor of ERK aggravated the 4-OHE2-induced cytotoxicity, supporting the pivotal role of ERK in protecting against catechol estrogen-induced oxidative cell death.

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

    SciTech Connect

    Garbe, James C.; Vrba, Lukas; Sputova, Klara; Fuchs, Laura; Novak, Petr; Brothman, Arthur R.; Jackson, Mark; Chin, Koei; LaBarge, Mark A.; Watts, George; Futscher, Bernard W.; Stampfer, Martha R.

    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 agents 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.

  5. 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

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

    PubMed Central

    Garbe, James C; Vrba, Lukas; Sputova, Klara; Fuchs, Laura; Novak, Petr; Brothman, Arthur R; Jackson, Mark; Chin, Koei; LaBarge, Mark A; Watts, George; Futscher, Bernard W; Stampfer, Martha R

    2014-01-01

    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 agents 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. PMID:25485586

  7. Isolation of canine mammary cells with stem cell properties and tumour-initiating potential.

    PubMed

    Cocola, C; Anastasi, P; Astigiano, S; Piscitelli, E; Pelucchi, P; Vilardo, L; Bertoli, G; Beccaglia, M; Veronesi, M C; Sanzone, S; Barbieri, O; Reinbold, R A; Luvoni, G C; Zucchi, I

    2009-07-01

    Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.

  8. Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme.

    PubMed

    Cunha, G R; Young, P; Christov, K; Guzman, R; Nandi, S; Talamantes, F; Thordarson, G

    1995-01-01

    The goal of this research was to establish methods for inducing mammary epithelial differentiation from nonmammary epithelium. For this purpose, mid-ventral or dorsal epidermis (skin epithelium; SKE) from 13-day rat or mouse embryos was associated with 13-day embryonic mouse mammary mesenchyme (mammary gland mesenchyme; MGM) (mouse MGM+rat or mouse SKE). The resultant MGM+SKE recombinants as well as controls (homotypic mouse mammary recombinants, homotypic mouse skin recombinants and mouse mammary mesenchyme by itself) were grafted under the renal capsule of syngeneic or athymic female nude mouse hosts. Most female hosts were induced to undergo lactogenesis by grafting an adult pituitary which elicited a state of hyperprolactinemia. Tissue recombinants of mouse MGM+rat or mouse SKE grown for 1 month in vivo formed a hair-bearing keratinized skin from which mammary ductal structures extended into the mesenchyme. The ducts were composed of columnar luminal epithelial cells as well as basal, actin-positive myoepithelial cells. When grown in pituitary-grafted hosts, the ductal epithelial cells expressed casein and alpha-lactalbumin as judged by immunocytochemistry. The expression of caseins in MGM+SKE recombinants was confirmed by Western blot. The epithelial cells in mouse MGM+rat SKE recombinants expressing milk proteins were shown to be rat cells while the surrounding connective tissue was composed of mouse cells based upon staining with Hoechst dye 33258. Using mammary-specific markers, these studies confirmed the earlier morphological studies of Propper and unequivocally demonstrated for the first time that embryonic mammary mesenchyme can induce morphological and functional mammary differentiation from nonmammary epithelium.

  9. Stem cells in normal mammary gland and breast cancer.

    PubMed

    Luo, Jie; Yin, Xin; Ma, Tao; Lu, Jun

    2010-04-01

    The mammary gland is a structurally dynamic organ that undergoes dramatic alterations with age, menstrual cycle, and reproductive status. Mammary gland stem cells, the minor cell population within the mature organ, are thought to have multiple functions in regulating mammary gland development, tissue maintenance, major growth, and structural remodeling. In addition, accumulative evidence suggests that breast cancers are initiated and maintained by a subpopulation of tumor cells with stem cell features (called cancer stem cells). A variety of methods have been developed to identify and characterize mammary stem cells, and several signal transduction pathways have been identified to be essential for the self-renewal and differentiation of mammary gland stem cells. Understanding the origin of breast cancer stem cells, their relationship to breast cancer development, and the differences between normal and cancer stem cells may lead to novel approaches to breast cancer diagnosis, prevention, and treatment.

  10. Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

    DTIC Science & Technology

    2007-06-01

    considered crucial for human carcinogenesis, in order for a single cell to accumulate the multiple errors necessary for malignancy. In human...prior to encountering stasis. Multiple types of single changes that prevent Rb-mediated growth inhibition will overcome stasis. Loss of CDKN2A...stringent barrier to human cellular immor- talization; in post-selection HMEC multiple errors appear to be necessary for telomerase reactivation, and

  11. CELL CONTACTS IN THE MOUSE MAMMARY GLAND

    PubMed Central

    Pitelka, Dorothy R.; Hamamoto, Susan T.; Duafala, Joan G.; Nemanic, Michael K.

    1973-01-01

    The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both. PMID:4569313

  12. Aurora kinase-A overexpression in mouse mammary epithelium induces mammary adenocarcinomas harboring genetic alterations shared with human breast cancer.

    PubMed

    Treekitkarnmongkol, Warapen; Katayama, Hiroshi; Kai, Kazuharu; Sasai, Kaori; Jones, Jennifer Carter; Wang, Jing; Shen, Li; Sahin, Aysegul A; Gagea, Mihai; Ueno, Naoto T; Creighton, Chad J; Sen, Subrata

    2016-12-01

    Recent data from The Cancer Genome Atlas analysis have revealed that Aurora kinase A (AURKA) amplification and overexpression characterize a distinct subset of human tumors across multiple cancer types. Although elevated expression of AURKA has been shown to induce oncogenic phenotypes in cells in vitro, findings from transgenic mouse models of Aurora-A overexpression in mammary glands have been distinct depending on the models generated. In the present study, we report that prolonged overexpression of AURKA transgene in mammary epithelium driven by ovine β-lactoglobulin promoter, activated through multiple pregnancy and lactation cycles, results in the development of mammary adenocarcinomas with alterations in cancer-relevant genes and epithelial-to-mesenchymal transition. The tumor incidence was 38.9% (7/18) in Aurora-A transgenic mice at 16 months of age following 4-5 pregnancy cycles. Aurora-A overexpression in the tumor tissues accompanied activation of Akt, elevation of Cyclin D1, Tpx2 and Plk1 along with downregulation of ERα and p53 proteins, albeit at varying levels. Microarray comparative genomic hybridization (CGH) analyses of transgenic mouse mammary adenocarcinomas revealed copy gain of Glp1r and losses of Ercc5, Pten and Tcf7l2 loci. Review of human breast tumor transcriptomic data sets showed association of these genes at varying levels with Aurora-A gain of function alterations. Whole exome sequencing of the mouse tumors also identified gene mutations detected in Aurora-A overexpressing human breast cancers. Our findings demonstrate that prolonged overexpression of Aurora-A can be a driver somatic genetic event in mammary adenocarcinomas associated with deregulated tumor-relevant pathways in the Aurora-A subset of human breast cancer.

  13. Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells.

    PubMed

    Vares, Guillaume; Sai, Sei; Wang, Bing; Fujimori, Akira; Nenoi, Mitsuru; Nakajima, Tetsuo

    2015-07-01

    Ionizing radiation and cumulative exposure to steroid hormones are known risk factors for breast cancer. There is increasing evidence that breast tumors are driven by a subpopulation of tumor-initiating cancer stem cells (CSCs). In MCF10A non-cancerous basal-like PR(-) cells, progesterone treatment and X-rays generated ALDH(+) and CD44(+)/CD24(-) CSCs. Here, we report that in irradiated MCF10A cells, progesterone activated the PI3K/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the PI3K/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of PI3K/Akt via mPR resulted in the inactivation of FOXO transcriptional activity, the upregulation of snail and slug expression and a downregulation of miR-29 expression, which led to increased levels of KLF4, a transcription factor required for breast CSC maintenance. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk in basal cells. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential novel anti-CSC strategies.

  14. Karyotypic instability and centrosome aberrations in the progeny of finite life-span human mammary epithelial cells exposed to sparsely or densely ionizing radiation.

    PubMed

    Sudo, Hiroko; Garbe, James; Stampfer, Martha R; Barcellos-Hoff, Mary Helen; Kronenberg, Amy

    2008-07-01

    The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer.

  15. A Comparative Approach of Tumor-Associated Inflammation in Mammary Cancer between Humans and Dogs.

    PubMed

    Carvalho, Maria Isabel; Silva-Carvalho, Ricardo; Pires, Isabel; Prada, Justina; Bianchini, Rodolfo; Jensen-Jarolim, Erika; Queiroga, Felisbina L

    2016-01-01

    Infiltrating cells of the immune system are widely accepted to be generic constituents of tumor microenvironment. It has been well established that the development of mammary cancer, both in humans and in dogs, is associated with alterations in numbers and functions of immune cells at the sites of tumor progression. These tumor infiltrating immune cells seem to exhibit exclusive phenotypic and functional characteristics and mammary cancer cells can take advantage of signaling molecules released by them. Cancer related inflammation has an important role in mammary carcinogenesis, contributing to the acquisition of core hallmark capabilities that allow cancer cells to survive, proliferate, and disseminate. Indeed, recent studies in human breast cancer and in canine mammary tumors have identified a growing list of signaling molecules released by inflammatory cells that serve as effectors of their tumor-promoting actions. These include the COX-2, the tumor EGF, the angiogenic VEGF, other proangiogenic factors, and a large variety of chemokines and cytokines that amplify the inflammatory state. This review describes the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in both human and dog mammary carcinogenesis.

  16. A Comparative Approach of Tumor-Associated Inflammation in Mammary Cancer between Humans and Dogs

    PubMed Central

    Silva-Carvalho, Ricardo; Pires, Isabel; Bianchini, Rodolfo; Jensen-Jarolim, Erika

    2016-01-01

    Infiltrating cells of the immune system are widely accepted to be generic constituents of tumor microenvironment. It has been well established that the development of mammary cancer, both in humans and in dogs, is associated with alterations in numbers and functions of immune cells at the sites of tumor progression. These tumor infiltrating immune cells seem to exhibit exclusive phenotypic and functional characteristics and mammary cancer cells can take advantage of signaling molecules released by them. Cancer related inflammation has an important role in mammary carcinogenesis, contributing to the acquisition of core hallmark capabilities that allow cancer cells to survive, proliferate, and disseminate. Indeed, recent studies in human breast cancer and in canine mammary tumors have identified a growing list of signaling molecules released by inflammatory cells that serve as effectors of their tumor-promoting actions. These include the COX-2, the tumor EGF, the angiogenic VEGF, other proangiogenic factors, and a large variety of chemokines and cytokines that amplify the inflammatory state. This review describes the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in both human and dog mammary carcinogenesis. PMID:28053982

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

    PubMed

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

    2011-06-01

    The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.

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

    PubMed

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

    2016-01-01

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

  19. Mouse mammary tumor virus suppresses apoptosis of mammary epithelial cells through ITAM-mediated signaling.

    PubMed

    Kim, Hyoung H; Grande, Shannon M; Monroe, John G; Ross, Susan R

    2012-12-01

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

  20. Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro.

    PubMed Central

    Bracke, M. E.; Vyncke, B. M.; Bruyneel, E. A.; Vermeulen, S. J.; De Bruyne, G. K.; Van Larebeke, N. A.; Vleminckx, K.; Van Roy, F. M.; Mareel, M. M.

    1993-01-01

    The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells. Images Figure 1 Figure 3 Figure 4 Figure 7 Figure 8 Figure 10 PMID:8347483

  1. 4-Hydroxyestradiol induces oxidative stress and apoptosis in human mammary epithelial cells: possible protection by NF-{kappa}B and ERK/MAPK

    SciTech Connect

    Chen Zhihua; Na, Hye-Kyung; Hurh, Yeon-Jin; Surh, Young-Joon . E-mail: surh@plaza.snu.ac.kr

    2005-10-01

    Catechol estrogens, the hydroxylated metabolites of 17{beta}-estradiol (E{sub 2}), have been considered to be implicated in estrogen-induced carcinogenesis. 4-Hydroxyestradiol (4-OHE{sub 2}), an oxidized metabolite of E{sub 2} formed preferentially by cytochrome P450 1B1, reacts with DNA to form depurinating adducts thereby exerting genotoxicity and carcinogenicity. 4-OHE{sub 2} undergoes 2-electron oxidation to quinone via semiquinone, and during this process, reactive oxygen species (ROS) can be generated to cause DNA damage and cell death. In the present study, 4-OHE{sub 2} was found to elicit cytotoxicity in cultured human mammary epithelial (MCF-10A) cells, which was blocked by the antioxidant trolox. MCF-10A cells treated with 4-OHE{sub 2} exhibited increased intracellular ROS accumulation and 8-oxo-7,8-dihydroxy-2'-deoxyguanosine formation, and underwent apoptosis as determined by poly(ADP-ribose)polymerase cleavage and disruption of mitochondrial transmembrane potential. The redox-sensitive transcription factor nuclear factor {kappa}B (NF-{kappa}B) was transiently activated by 4-OHE{sub 2} treatment. Cotreatment of MCF-10A cells with the NF-{kappa}B inhibitor, L-1-tosylamido-2-phenylethyl chloromethyl ketone, exacerbated 4-OHE{sub 2}-induced cell death. 4-OHE{sub 2} also caused transient activation of extracellular signal-regulated protein kinases (ERK) involved in transmitting cell survival or death signals. A pharmacological inhibitor of ERK aggravated the 4-OHE{sub 2}-induced cytotoxicity, supporting the pivotal role of ERK in protecting against catechol estrogen-induced oxidative cell death.

  2. Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCAl Genes

    DTIC Science & Technology

    1997-10-01

    We originally proposed the Differential Display method (Liang and Pardee 1992) to identify genes that are modulated by p53 and BRCA1 deficiency. As...retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc Natl Acad Sci U S A 93:5185-5190 (1996). Liang P, Pardee A...Shoemaker A, Dove W. ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer 3 1A: 1061-4 (1995). Moser A, Mattes E, Dove W, Lindstrom M

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

    DTIC Science & Technology

    2013-10-01

    most recent funding period, our bioinformatic analysis identified subsets of fMaSC signature genes that are coordinately expressed in archived human...adapted a new microfluidics -based, single-cell capture and library preparation system to improve reproducibility in the generation of gene expression...fraction, a two-class Significance Analysis of Microarrays (SAM) [7, 8] was performed within each dataset comparing each given FACS population

  4. Mammary gland stem cells and their application in breast cancer.

    PubMed

    Yang, Xing; Wang, Hui; Jiao, Baowei

    2017-02-07

    The mammary gland is an organ comprising two primary lineages, specifically the inner luminal and the outer myoepithelial cell layers. Mammary gland stem cells (MaSCs) are highly dynamic and self-renewing, and can give rise to these mammary gland lineages. The lineages are responsible for gland generation during puberty as well as expansion during pregnancy. In recent years, researchers have focused on understanding how MaSCs are regulated during mammary gland development and transformation of breast cancer. Here, we summarize the identification of MaSCs, and how they are regulated by the signaling transduction pathways, mammary gland microenvironment, and non-coding RNAs (ncRNAs). Moreover, we debate the evidence for their serving as the origin of breast cancer, and discuss the therapeutic perspectives of targeting breast cancer stem cells (BCSCs). In conclusion, a better understanding of the key regulators of MaSCs is crucial for the clinical treatment of breast cancer.

  5. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

    PubMed Central

    Asch, B B; Burstein, N A; Vidrich, A; Sun, T T

    1981-01-01

    Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression. Images PMID:6170984

  6. Developmental biology: cell fate in the mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Serotonin suppresses β-casein expression via inhibition of the signal transducer and activator of transcription 5 (STAT5) protein phosphorylation in human mammary epithelial cells MCF-12A.

    PubMed

    Chiba, Takeshi; Kimura, Soichiro; Takahashi, Katsuo; Morimoto, Yasunori; Sanbe, Atsushi; Ueda, Hideo; Kudo, Kenzo

    2014-01-01

    Serotonin (5-hydroxytryptamine; 5-HT) has an important physiological role in controlling lactation, namely, milk volume homeostasis, within mammary glands. The objectives of this study were to evaluate whether exogenous 5-HT can suppress β-casein expression, a differentiation marker, produced in human mammary epithelial cells, and to determine whether 5-HT can attenuate β-casein signaling via the prolactin (PRL) receptor (PRLr) and Janus kinase 2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL treatment increased the mRNA level of β-casein in the MCF-12A human mammary epithelial cell line, and the highest level occurred at days 7 and 14 of culture. In contrast, PRLr expression was not affected significantly by PRL treatment. PRL treatment in MCF-12A cells increased levels of β-casein and phosphorylated STAT5 (pSTAT5) proteins in a concentration-dependent manner, with a slight increase of STAT5 protein. β-Casein expression was inhibited by 0.1 mM 5-HT in a time-dependent manner. Additionally, treatment with 0.1 mM 5-HT for 72 h decreased protein levels of β-casein and pSTAT5, with a slight decrease in STAT5 levels. These results suggest that exogenous 5-HT can inhibit STAT5 phosphorylation, resulting in a decrease in β-Casein expression. In conclusion, we showed that exogenous 5-HT decreased β-casein expression in MCF-12A human mammary epithelial cells, and that 5-HT was responsible for inhibiting phosphorylation of STAT5, resulting in a decline in lactational function.

  8. Cell hierarchy and lineage commitment in the bovine mammary gland.

    PubMed

    Rauner, Gat; Barash, Itamar

    2012-01-01

    The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin⁻ epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24(med)CD49f(pos) (putative stem cells, puStm), CD24(neg)CD49f(pos) (Basal), CD24(high)CD49f(neg) (putative progenitors, puPgt) and CD24(med)CD49f(neg) (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell-enriched.

  9. Snapshots of mammary gland interstitial cells: methylene-blue vital staining and c-kit immunopositivity.

    PubMed

    Popescu, L M; Andrei, F; Hinescu, M E

    2005-01-01

    We show here that methylene-blue supravital staining of specimens from normal human mammary gland reveals (selectively) interstitial (stromal) cells, with 2-3 long (20-80 microm), thin, moniliform processes. Such cells appear c-kit/CD117 positive, either by immunohistochemistry (IHC) or immunofluorescence (IF). Since these features (affinity for methylene blue, c-kit positivity, and characteristic processes) define archetypal interstitial cells of Cajal (ICC) in light microscopy, our results suggest the existence of Cajal-like cells in the interstitium of human normal mammary gland.

  10. Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2

    PubMed Central

    2014-01-01

    Introduction The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. Methods FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. Results Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. Conclusions Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity. PMID:24451168

  11. Immune response signatures of benzo{alpha}pyrene exposure in normal human mammary epithelial cells in the absence or presence of chlorophyllin.

    PubMed

    John, Kaarthik; Keshava, Channa; Richardson, Diana L; Weston, Ainsley; Nath, Joginder

    2009-01-01

    Carcinogenic polycylic aromatic hydrocarbons can alter immune responses. Changes in immune response gene expression profiles in multiple human mammary cell strains exposed to benzo(alpha)pyrene (BP) (4 microM) in vitro, in the presence or absence of chlorophyllin (5 microM), were observed using Affymetrix gene arrays. Expressions of five immune response genes were altered ~3.0-fold by BP exposure and 24 genes by BP in the presence chlorophyllin. In silico pathway analysis revealed altered immune response genes form interactive gene networks with many cellular processes, suggesting their role in a complex multigenic response to toxins. Additionally, it was suggestive of the possible immunomodulatory potential of chlorophyllin apart from various other well-documented mechanisms of action. Gene expression matrices revealed consistent alteration patterns involving IL1B, SECTM1 and CXCL14 on exposure to BP, and IL1RN, CD86, IF144 and GIP2 in the presence of chlorophyllin and BP, suggesting some of these genes might constitute putative immune response biomarkers of PAH exposure. This study has therefore identified a battery of potential immune response biomarkers of PAH exposure, amidst several genes, for future validation studies.

  12. 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.

  13. Immune cell location and function during post-natal mammary gland development.

    PubMed

    Reed, Johanna R; Schwertfeger, Kathryn L

    2010-09-01

    Post-natal mammary gland development requires complex interactions between the epithelial cells and various cell types within the stroma. Recent studies have illustrated the importance of immune cells and their mediators during the various stages of mammary gland development. However, the mechanisms by which these immune cells functionally contribute to mammary gland development are only beginning to be understood. This review provides an overview of the localization of immune cells within the mammary gland during the various stages of post-natal mammary gland development. Furthermore, recent studies are summarized that illustrate the mechanisms by which these cells are recruited to the mammary gland and their functional roles in mammary gland development.

  14. Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay.

    PubMed

    Deman, J J; Van Larebeke, N A; Bruyneel, E A; Bracke, M E; Vermeulen, S J; Vennekens, K M; Mareel, M M

    1995-09-01

    MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

  15. 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.

  16. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

  17. Identification and characterization of cancer stem cells in canine mammary tumors.

    PubMed

    Rybicka, Agata; Król, Magdalena

    2016-12-19

    Cancer stem cells (CSC) represent a small subpopulation of cells in malignant tumors that possess the unique ability to self-renew, differentiate and resist chemo- and radiotherapy. These cells have been postulated to be the basis for some of the difficulties in treating cancer, and therefore, numerous approaches have been developed to specifically target and eliminate CSC in diverse types of cancer, including breast cancer. Spontaneously occurring mammary tumors in canines share clinical and molecular similarities with the human counterpart, making the dog a potentially powerful model for the study of human breast cancer and clinical trials. Studies focused on canine mammary CSC might therefore enhance our understanding of the biology and possible treatment of the disease in both dogs and humans. In this review, we discuss various approaches currently in use to isolate and characterize canine mammary CSC.

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

    SciTech Connect

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

    2014-10-02

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

  19. Differential gene expression pattern in human mammary epithelial cells induced by realistic organochlorine mixtures described in healthy women and in women diagnosed with breast cancer.

    PubMed

    Rivero, Javier; Henríquez-Hernández, Luis Alberto; Luzardo, Octavio P; Pestano, José; Zumbado, Manuel; Boada, Luis D; Valerón, Pilar F

    2016-03-30

    Organochlorine pesticides (OCs) have been associated with breast cancer development and progression, but the mechanisms underlying this phenomenon are not well known. In this work, we evaluated the effects exerted on normal human mammary epithelial cells (HMEC) by the OC mixtures most frequently detected in healthy women (H-mixture) and in women diagnosed with breast cancer (BC-mixture), as identified in a previous case-control study developed in Spain. Cytotoxicity and gene expression profile of human kinases (n=68) and non-kinases (n=26) were tested at concentrations similar to those described in the serum of those cases and controls. Although both mixtures caused a down-regulation of genes involved in the ATP binding process, our results clearly indicate that both mixtures may exert a very different effect on the gene expression profile of HMEC. Thus, while BC-mixture up-regulated the expression of oncogenes associated to breast cancer (GFRA1 and BHLHB8), the H-mixture down-regulated the expression of tumor suppressor genes (EPHA4 and EPHB2). Our results indicate that the composition of the OC mixture could play a role in the initiation processes of breast cancer. In addition, the present results suggest that subtle changes in the composition and levels of pollutants involved in environmentally relevant mixtures might induce very different biological effects, which explain, at least partially, why some mixtures seem to be more carcinogenic than others. Nonetheless, our findings confirm that environmentally relevant pollutants may modulate the expression of genes closely related to carcinogenic processes in the breast, reinforcing the role exerted by environment in the regulation of genes involved in breast carcinogenesis.

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

    DOE PAGES

    Gong, Chunhong; Zhang, Yi; Shankaran, Harish; ...

    2014-10-02

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Mammary extracellular matrix directs differentiation of testicular and embryonic stem cells to form functional mammary glands in vivo

    PubMed Central

    Bruno, Robert D.; Fleming, Jodie M.; George, Andrea L.; Boulanger, Corinne A.; Schedin, Pepper; Smith, Gilbert H.

    2017-01-01

    Previously, we demonstrated the ability of the normal mammary microenvironment (niche) to direct non-mammary cells including testicular and embryonic stem cells (ESCs) to adopt a mammary epithelial cell (MEC) fate. These studies relied upon the interaction of transplanted normal MECs with non-mammary cells within the mammary fat-pads of recipient mice that had their endogenous epithelium removed. Here, we tested whether acellular mammary extracellular matrix (mECM) preparations are sufficient to direct differentiation of testicular-derived cells and ESCs to form functional mammary epithelial trees in vivo. We found that mECMs isolated from adult mice and rats were sufficient to redirect testicular derived cells to produce normal mammary epithelial trees within epithelial divested mouse mammary fat-pads. Conversely, ECMs isolated from omental fat and lung did not redirect testicular cells to a MEC fate, indicating the necessity of tissue specific components of the mECM. mECM preparations also completely inhibited teratoma formation from ESC inoculations. Further, a phenotypically normal ductal outgrowth resulted from a single inoculation of ESCs and mECM. To the best of our knowledge, this is the first demonstration of a tissue specific ECM driving differentiation of cells to form a functional tissue in vivo. PMID:28071703

  3. SERMs attenuate estrogen-induced malignant transformation of human mammary epithelial cells by upregulating detoxification of oxidative metabolites

    PubMed Central

    Madhubhani, L.P.; Hemachandra, P.; Patel, Hitisha; Esala, R.; Chandrasena, P.; Choi, Jaewoo; Piyankarage, Sujeewa C.; Wang, Shuai; Wang, Yijin; Thayer, Emily; Scism, Rob; Michalsen, Bradley T.; Xiong, Rui; Siklos, Marton; Bolton, Judy L.; Thatcher, Gregory R.J.

    2014-01-01

    The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER), and chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Non-tumorigenic MCF-10A human breast epithelial cells are classified as ER(−) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERMs), in use for breast cancer chemoprevention and for post-menopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular ROS, and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of Phase 2 rather than Phase 1 metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of Phase 2 metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs. PMID:24598415

  4. Long-term exposure to bisphenol A or benzo(a)pyrene alters the fate of human mammary epithelial stem cells in response to BMP2 and BMP4, by pre-activating BMP signaling

    PubMed Central

    Clément, Flora; Xu, Xinyi; Donini, Caterina F; Clément, Alice; Omarjee, Soleilmane; Delay, Emmanuel; Treilleux, Isabelle; Fervers, Béatrice; Le Romancer, Muriel; Cohen, Pascale A; Maguer-Satta, Véronique

    2017-01-01

    Bone morphogenetic protein 2 (BMP2) and BMP4 are key regulators of the fate and differentiation of human mammary epithelial stem cells (SCs), as well as of their niches, and are involved in breast cancer development. We established that MCF10A immature mammary epithelial cells reliably reproduce the BMP response that we previously identified in human primary epithelial SCs. In this model, we observed that BMP2 promotes luminal progenitor commitment and expansion, whereas BMP4 prevents lineage differentiation. Environmental pollutants are known to promote cancer development, possibly by providing cells with stem-like features and by modifying their niches. Bisphenols, in particular, were shown to increase the risk of developing breast cancer. Here, we demonstrate that chronic exposure to low doses of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) alone has little effect on SCs properties of MCF10A cells. Conversely, we show that this exposure affects the response of immature epithelial cells to BMP2 and BMP4. Furthermore, the modifications triggered in MCF10A cells on exposure to pollutants appeared to be predominantly mediated by altering the expression and localization of type-1 receptors and by pre-activating BMP signaling, through the phosphorylation of small mothers against decapentaplegic 1/5/8 (SMAD1/5/8). By analyzing stem and progenitor properties, we reveal that BPA prevents the maintenance of SC features prompted by BMP4, whereas promoting cell differentiation towards a myoepithelial phenotype. Inversely, B(a)P prevents BMP2-mediated luminal progenitor commitment and expansion, leading to the retention of stem-like properties. Overall, our data indicate that BPA and B(a)P distinctly alter the fate and differentiation potential of mammary epithelial SCs by modulating BMP signaling. PMID:27740625

  5. Attenuation of TGF-β signaling suppresses premature senescence in a p21-dependent manner and promotes oncogenic Ras-mediated metastatic transformation in human mammary epithelial cells.

    PubMed

    Lin, Shu; Yang, Junhua; Elkahloun, Abdel G; Bandyopadhyay, Abhik; Wang, Long; Cornell, John E; Yeh, I-Tien; Agyin, Joseph; Tomlinson, Gail; Sun, Lu-Zhe

    2012-04-01

    The molecular mechanisms that drive triple-negative, basal-like breast cancer progression are elusive. Few molecular targets have been identified for the prevention or treatment of this disease. Here we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered transforming growth factor-β (TGF-β) sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative type II receptor (DNRII) of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-Ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, coexpression of DNRII and H-Ras-V12 rendered HMECs highly tumorigenic and metastatic in vivo in comparison with H-Ras-V12-transformed HMECs that spontaneously escaped H-Ras-V12-induced SLGA. Microarray analysis revealed that p21 was the major player mediating Ras-induced SLGA, and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling was blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-Ras-V12-induced SLGA. Our results identify that autocrine TGF-β signaling is an integral part of the cellular anti-transformation network by suppressing the expression of a host of genes, including p21-regulated genes, that mediate oncogene-induced transformation in basal-like breast cancer.

  6. Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway.

    PubMed

    Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke; Takahashi, Tatsuyuki; Obara, Yoshiaki

    2006-12-11

    Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.

  7. Mammary gland development: cell fate specification, stem cells and the microenvironment.

    PubMed

    Inman, Jamie L; Robertson, Claire; Mott, Joni D; Bissell, Mina J

    2015-03-15

    The development of the mammary gland is unique: the final stages of development occur postnatally at puberty under the influence of hormonal cues. Furthermore, during the life of the female, the mammary gland can undergo many rounds of expansion and proliferation. The mammary gland thus provides an excellent model for studying the 'stem/progenitor' cells that allow this repeated expansion and renewal. In this Review, we provide an overview of the different cell types that constitute the mammary gland, and discuss how these cell types arise and differentiate. As cellular differentiation cannot occur without proper signals, we also describe how the tissue microenvironment influences mammary gland development.

  8. Mast cells in canine cutaneous hemangioma, hemangiosarcoma and mammary tumors.

    PubMed

    Woldemeskel, Moges; Rajeev, Sreekumari

    2010-02-01

    Mast cell count (MCC) in 45 dogs with cutaneous hemangioma (HA, n = 12), hemangiosarcoma (HSA, n = 12), mammary adenoma (AD, n = 9) and mammary adenocarcinoma (AC, n = 12) was made using Toluidine blue stained sections. Antibodies against endothelial cell markers, Factor VIII and VEGF were used to visualize and determine the hot spot micro-vessel density (MVD). Total MCC and MCC along the invasive edges were significantly higher (p < 0.001) in canine mammary AC than in AD. The total MCC did not significantly differ (p > 0.05), in HSAs (8.6 +/- 3.3) than in HAs (5.5 +/- 2.8). There is a positive correlation (r = 0.14) between the hot spot MCC and MVD in mammary AC, although not significant (p = 0.3172), indicating that mast cells are associated with angiogenesis in canine mammary AC. This study suggests that mast cells may play an important role in neovascularization of canine cutaneous vascular and mammary neoplasms. Detailed studies encompassing correlation of MCC and MVD with clinical outcomes and prognosis in these neoplasms are recommended.

  9. Precursors of hexoneogenesis within the human mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Comparative expression pathway analysis of human and canine mammary tumors

    PubMed Central

    Uva, Paolo; Aurisicchio, Luigi; Watters, James; Loboda, Andrey; Kulkarni, Amit; Castle, John; Palombo, Fabio; Viti, Valentina; Mesiti, Giuseppe; Zappulli, Valentina; Marconato, Laura; Abramo, Francesca; Ciliberto, Gennaro; Lahm, Armin; La Monica, Nicola; de Rinaldis, Emanuele

    2009-01-01

    Background Spontaneous tumors in dog have been demonstrated to share many features with their human counterparts, including relevant molecular targets, histological appearance, genetics, biological behavior and response to conventional treatments. Mammary tumors in dog therefore provide an attractive alternative to more classical mouse models, such as transgenics or xenografts, where the tumour is artificially induced. To assess the extent to which dog tumors represent clinically significant human phenotypes, we performed the first genome-wide comparative analysis of transcriptional changes occurring in mammary tumors of the two species, with particular focus on the molecular pathways involved. Results We analyzed human and dog gene expression data derived from both tumor and normal mammary samples. By analyzing the expression levels of about ten thousand dog/human orthologous genes we observed a significant overlap of genes deregulated in the mammary tumor samples, as compared to their normal counterparts. Pathway analysis of gene expression data revealed a great degree of similarity in the perturbation of many cancer-related pathways, including the 'PI3K/AKT', 'KRAS', 'PTEN', 'WNT-beta catenin' and 'MAPK cascade'. Moreover, we show that the transcriptional relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Conclusion Our data confirm and further strengthen the value of the canine mammary cancer model and open up new perspectives for the evaluation of novel cancer therapeutics and the development of prognostic and diagnostic biomarkers to be used in clinical studies. PMID:19327144

  11. Autophagy regulates keratin 8 homeostasis in mammary epithelial cells and in breast tumors

    PubMed Central

    Kongara, Sameera; Kravchuk, Olga; Teplova, Irina; Lozy, Fred; Schulte, Jennifer; Moore, Dirk; Barnard, Nicola; Neumann, Carola A.; White, Eileen; Karantza, Vassiliki

    2010-01-01

    Autophagy is activated in response to cellular stressors and mediates lysosomal degradation and recycling of cytoplasmic material and organelles as a temporary cell survival mechanism. Defective autophagy is implicated in human pathology, as disruption of protein and organelle homeostasis enables disease-promoting mechanisms such as toxic protein aggregation, oxidative stress, genomic damage and inflammation. We previously showed that autophagy-defective immortalized mouse mammary epithelial cells (iMMECs) are susceptible to metabolic stress, DNA damage and genomic instability. We now report that autophagy deficiency was associated with ER and oxidative stress, and deregulation of p62-mediated keratin homeostasis in mammary cells and allograft tumors and in mammary tissues from genetically engineered mice. In human breast tumors, high phospho(Ser73)-K8 levels inversely correlated with Beclin 1 expression. Thus, autophagy preserves cellular fitness by limiting ER and oxidative stress, a function potentially important in autophagy-mediated suppression of mammary tumorigenesis. Furthermore, autophagy regulates keratin homeostasis in the mammary gland via a p62-dependent mechanism. High phospho(Ser73)-K8 expression may be a marker of autophagy functional status in breast tumors and, as such, could have therapeutic implications for breast cancer patients. PMID:20530580

  12. 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

    transcriptomic data. 4. Genetic strategies were employed to show that SOX10 is required for fMaSC function in vitro and in vivo. 5. Developed a genetic ...Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts. Genome Biol. 14(11):R125 epub

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

    DTIC Science & Technology

    2013-10-01

    bioinformatic analysis identified subsets of fMaSC signature genes that are coordinately expressed in archived human breast cancer gene expression data sets and...existing and commonly used clinical variables in the preliminary data sets we have analyzed. We have also adapted a new microfluidics -based, single... Analysis of Microarrays (SAM) [7, 8] was performed within each dataset comparing each given FACS population versus all others from that experimental

  14. Effects of several dioxin-like compounds on estrogen metabolism in the malignant MCF-7 and nontumorigenic MCF-10A human mammary epithelial cell lines.

    PubMed

    van Duursen, Majorie B M; Sanderson, J Thomas; van der Bruggen, Marieke; van der Linden, Jeroen; van den Berg, Martin

    2003-08-01

    In human breast tissue, estrone (E(1)) and estradiol (E(2)) are mainly hydroxylated by cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) to 2-hydroxyestrogens (2-OHE(1/2)) and 4-hydroxyestrogens (4-OHE(1/2)), respectively. Several studies show that 4-OHE(1/2), but not 2-OHE(1/2), may act as a carcinogen and a high estrogen 4-/2-hydroxylation ratio appears to be a marker for the presence of neoplasms. In this study, we investigated the effects of several dioxin-like compounds on estrogen 2- and 4-hydroxylation in a malignant (MCF-7) and a nontumorigenic (MCF-10A) human mammary epithelial cell line. 2- and 4-methoxyestrogen (MeOE(1/2)) formations were used as measures of the 2- and 4-hydroxylation pathways, respectively. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,3'4,4',5,5'-hexachlorobiphenyl (PCB 169) concentration dependently induced 2-MeOE(1/2) formation and ethoxyresorufin-O-deethylation (EROD) activity through induced CYP1A1 expression in MCF-7 and MCF-10A cells. 2,3',4,4',5-pentachlorobiphenyl (PCB 118) had no such effect. Effects on CYP1B1 expression and 4-MeOE(1/2) formation were less pronounced; only TCDD caused an induction, whereas PCB 169 was a potent and selective inhibitor of 4-MeOE(1/2) formation (IC(50) 0.7 and 2.2 nM PCB 169 in MCF-7 and MCF-10A cells, respectively). MCF-10A cells were less responsive toward dioxin-like compounds and the apparent EC(50) values for CYP1A1 and CYP1B1 induction in this study were 10-100 fold higher than in MCF-7 cells. The constitutive 4-/2-MeOE(1/2) ratios were 2.99 +/- 0.78 and 0.93 +/- 0.40 in MCF-7 and MCF-10A, respectively. Incubation with dioxin-like compounds resulted in a concentration-dependent decrease in the 4-/2-MeOE(1/2) ratio, but an increase in potentially carcinogenic estrogen metabolites in both MCF-7 and MCF-10A cells. This indicates that even though the 4-/2-OHE(1/2) ratio may be used as indicator for the presence

  15. Differential transformation of mammary epithelial cells by Wnt genes.

    PubMed Central

    Wong, G T; Gavin, B J; McMahon, A P

    1994-01-01

    The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent. Images PMID:8065359

  16. Identification of Novel Genes Affected by Gamma Irradiation Using a Gene-Trapped Library of Human Mammary Epithelial Cells

    DTIC Science & Technology

    2005-04-01

    Chromosomal and chromatid analysis was performed on the DREV 1 knockdown MCF10A cells to access cel l survival following ionizing radiation treatment...statement of work as well as their response to ionizing radiation . 14 . SUBJECT TERMS 15 . NUMBER OF PAGE S 3 0 Gamma Irradiation, gene trapping...line with and without ionizing radiation treatment . We felt that it was important t o analyze the identified gene expression levels following IR

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

    DTIC Science & Technology

    2006-08-01

    processes of mammary epithelial cell differentiation during developmentand tumorigenesis. Using FACS, mammary epithelial cell ( MEC ) populations from tumors...developed techniques for viral transduction andtransplantation of primary MECs . 15. SUBJECT TERMS mammary, stem cell, lentivirus, FACS, cancer, imaging 16...epithelial cell ( MEC ) populations from tumors and wildtype tissue was investigated for their outgrowth potential or tumorigenic capacity. We also developed

  18. 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.

  19. Parsing ERK Activation Reveals Quantitatively Equivalent Contributions From Epidermal Growth Factor Receptor and HER2 In Human Mammary Epithelial Cells

    SciTech Connect

    Hendriks, Bart S.; Orr, Galya; Wells, Alan H.; Wiley, H. S.; Lauffenburger, Douglas A.

    2005-02-18

    HER2, a member of the EGFR tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we apply a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2, and their downstream activation of extracellular signal-related kinase (ERK) to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we can separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrate that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activated ERK are quantitatively equivalent . We find that HER2-mediated effects on EGFR dimerization and trafficking are sufficient to explain the detected HER2-mediated amplification of EGF-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared to the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking, with resultant EGFR sparing, cause the sustained HER2-mediated enhancement of ERK signaling.

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

    PubMed

    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.

  1. Differential expression of living mammary epithelial cell subpopulations in milk during lactation in dairy cows.

    PubMed

    Baratta, M; Volpe, M G; Nucera, D; Gabai, G; Guzzo, N; Fustini, M; Faustini, M; Martignani, E

    2015-10-01

    Epithelial cells are shed into milk during lactation, and although they generally reflect the cellular characteristics of terminally differentiated luminal cells, previously the detection of more primitive cells was described in human milk where a cell population of epithelial lineage was detected expressing markers typical of progenitor cells. In this investigation, we report the development of flow cytometry analysis to allow multiparametric assessment of mammary epithelial cells observed in milk. Cells collected from milk samples of 10 healthy dairy cows were directly analyzed for 6 different markers: CD45, CD49f, cytokeratin 14, cytokeratin 18, presence of nucleus, and cell viability. Milk samples were collected in 3 different periods of lactation: early lactation (EL=d 0-30), mid-lactation (ML=d 90-120), and late lactation (LL=210-250). Here we identify the differential expression of precursor or differentiated cell markers (or both) in mammary epithelial cells present in bovine milk. Myoepithelial cells, as indicated by cells staining positively for cytokeratin 14(+)/cytokeratin 18(-), were observed to increase from EL to LL with a high correlation with nuclear staining inferring potential proliferative activity. Furthermore, a significant increase in CD49f(+) and cytokeratin 14(+)/cytokeratin 18(+) positive cells was observed in LL. This assay is a sensitive approach for evaluating the variations in the frequency and features of living epithelial cells, whose reciprocal balance may be significant in understanding mammary gland cellular function throughout lactation. These observations suggest that mammary epithelial cell immunophenotypes could be investigated as biomarkers for mammary gland function in dairy cows.

  2. Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines

    PubMed Central

    Kittrell, Frances; Valdez, Kelli; Elsarraj, Hanan; Hong, Yan; Medina, Daniel; Behbod, Fariba

    2016-01-01

    The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2–4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec). PMID:27446983

  3. Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts

    PubMed Central

    Boutinaud, Marion; Herve, Lucile; Lollivier, Vanessa

    2015-01-01

    Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants’ udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation). PMID:26579195

  4. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells

    SciTech Connect

    Brown, C.F.; Teng, C.T.; Pentecost, B.T.; DiAugustine, R.P. )

    1989-07-01

    Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-(35S)methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.

  5. Immunocytochemical Localization of Sex Steroid Hormone Receptors in Normal Human Mammary Gland

    PubMed Central

    Li, Sijie; Han, Bing; Liu, Guojin; Li, Songyun; Ouellet, Johanne; Labrie, Fernand; Pelletier, Georges

    2010-01-01

    The sex steroids, estrogens, progesterone, and androgens, all play a role in mammary development and function. To precisely identify the sites of action of these steroids, we studied the localization of the estrogen receptor α (ERα) and ERβ, the progesterone receptor A (PRA) and PRB, and androgen receptors (AR) in the normal human mammary gland. Immunocytochemical localization of ERα, ERβ, PRA, PRB, and AR was performed with reduction mammoplasty specimens from premenopausal women. ERα, PRA, PRB, and AR were localized mostly to the inner layer of epithelial cells lining acini and intralobular ducts, as well as to myoepithelial cells scattered in the external layer of interlobular ducts. AR was also found in some stromal cells. ERβ staining was more widespread, resulting in epithelial and myoepithelial cells being labeled in acini and ducts as well as stromal cells. These results suggest that all sex steroids can directly act on epithelial cells to modulate development and function of the human mammary gland. Estrogens and androgens can also indirectly influence epithelial cell activity by an action on stromal cells. (J Histochem Cytochem 58:509–515, 2010) PMID:20026671

  6. Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene

    PubMed Central

    Divi, Rao L.; Einem Lindeman, Tracey L.; Shockley, Marie E.; Keshava, Channa; Weston, Ainsley; Poirier, Miriam C.

    2014-01-01

    The polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) is thought to bind covalently to DNA, through metabolism by cytochrome P450 1A1 (CYP1A1) and CYP1B1, and other enzymes, to form r7, t8, t9-trihydroxy-c-10-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]-pyrene (BPdG). Evaluation of RNA expression data, to understand the contribution of different metabolic enzymes to BPdG formation, is typically presented as fold-change observed upon BP exposure, leaving the actual number of RNA transcripts unknown. Here, we have quantified RNA copies/ng cDNA (RNA cpn) for CYP1A1 and CYP1B1, as well as NAD(P)H:quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58–836 for CYP1A1, 336–5587 for CYP1B1 and 5943–40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251–13234 for CYP1A1, 4133–57078 for CYP1B1 and 4456–55887 for NQO1. There were 3.5 (mean, range 0.2–15.8) BPdG adducts/108 nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO

  7. Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue

    PubMed Central

    Moss, Stephanie C.; Bates, Michael; Parrino, Patrick E.; Woods, T. Cooper

    2007-01-01

    Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease. PMID:21603530

  8. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE 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 PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  9. Mammary Stem Cell Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

    PubMed Central

    Zhang, Zheng; Christin, John R.; Wang, Chunhui; Ge, Kai; Oktay, Maja H.; Guo, Wenjun

    2016-01-01

    SUMMARY Cancer genomics have provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic GEMMs (genetically engineered mouse models). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study established a robust in vivo platform for functional cancer genomics and discovered functional breast cancer mutations. PMID:27653681

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

    SciTech Connect

    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 (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 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{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.

  11. Sesamin synergistically potentiates the anticancer effects of γ-tocotrienol in mammary cancer cell lines.

    PubMed

    Akl, Mohamed R; Ayoub, Nehad M; Abuasal, Bilal S; Kaddoumi, Amal; Sylvester, Paul W

    2013-01-01

    γ-Tocotrienol and sesamin are phytochemicals that display potent anticancer activity. Since sesamin inhibits the metabolic degradation of tocotrienols, studies were conducted to determine if combined treatment with sesamin potentiates the antiproliferative effects of γ-tocotrienol on neoplastic mouse (+SA) and human (MCF-7 and MDA-MB-231) mammary cancer cells. Results showed that treatment with γ-tocotrienol or sesamin alone induced a significant dose-responsive growth inhibition, whereas combination treatment with these agents synergistically inhibited the growth of +SA, MCF-7 and MDA-MB-231 mammary cancer cells, while similar treatment doses were found to have little or no effect on normal (mouse CL-S1 and human MCF-10A) mammary epithelial cell growth or viability. However, sesamin synergistic enhancement of γ-tocotrienol-induced anticancer effects was not found to be mediated from a reduction in γ-tocotrienol metabolism. Rather, combined treatment with subeffective doses of γ-tocotrienol and sesamin was found to induce G1 cell cycle arrest, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6, phospho-Rb, and E2F1 levels, and increase in p27 and p16 levels. Additional studies showed that the antiproliferative effect of combination treatment did not initiate apoptosis or result in a decrease in mammary cancer cell viability. Taken together, these findings indicate that the synergistic antiproliferative action of combined γ-tocotrienol and sesamin treatment in mouse and human mammary cancer cells is cytostatic, not cytotoxic, and results from G1 cell cycle arrest.

  12. Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling

    PubMed Central

    Cucuzza, Laura Starvaggi; Motta, Massimiliano; Miretti, Silvia; Accornero, Paolo

    2008-01-01

    Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium. PMID:19116450

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

    SciTech Connect

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

    1997-10-13

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

  14. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  16. Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

    PubMed Central

    Chang, Hsiang-Hua D.; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J.; Pan, Yu; Zhou, Emily; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells. PMID:25786220

  17. Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

    PubMed

    Chang, Hsiang-Hua D; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J; Pan, Yu; Zhou, Emily; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

  18. Progesterone receptor gene maps to human chromosome band 11q13, the site of the mammary oncogene int-2

    SciTech Connect

    Law, M.L.; Kao, F.T.; Wei, Q.; Hartz, J.A.; Greene, G.L.; Zarucki-Schulz, T.; Conneely, O.M.; Jones, C.; Puck, T.T.; O'Malley, B.W.; Horwitz, K.B.

    1987-05-01

    Progesterone is involved in the development and progression of breast cancers, and progesterone receptors (PR) are important markers of hormone dependence and disease prognosis. The authors have used a human PR cDNA probe, genomic DNA blotting of a series of Chinese hamster-human cell hybrids, and in situ hybridization to map the human PR gene to chromosome 11, band q13. This band also contains the human homolog of the mouse mammary tumor virus integration site, int-2, which surrounds a protooncogene thought to be involved in the development of murine mammary cancers. That these two genes share the same chromosomal location raises important questions about their possible linkage and about the relationship between the mammary-specific oncogene and the steroid hormone in the development, growth, and hormone dependence of human breast cancers.

  19. Significance of rat mammary tumors for human risk assessment.

    PubMed

    Russo, Jose

    2015-02-01

    We have previously indicated that the ideal animal tumor model should mimic the human disease. This means that the investigator should be able to ascertain the influence of host factors on the initiation of tumorigenesis, mimic the susceptibility of tumor response based on age and reproductive history, and determine the response of the tumors induced to chemotherapy. The utilization of experimental models of mammary carcinogenesis in risk assessment requires that the influence of ovarian, pituitary, and placental hormones, among others, as well as overall reproductive events are taken into consideration, since they are important modifiers of the susceptibility of the organ to neoplastic development. Several species, such as rodents, dogs, cats, and monkeys, have been evaluated for these purposes; however, none of them fulfills all the criteria specified previously. Rodents, however, are the most widely used models; therefore, this work will concentrate on discussing the rat rodent model of mammary carcinogenesis.

  20. Screening and analysis of breast cancer genes regulated by the human mammary microenvironment in a humanized mouse model

    PubMed Central

    Zheng, Mingjie; Wang, Jue; Ling, Lijun; Xue, Dandan; Wang, Shui; Zhao, Yi

    2016-01-01

    Tumor microenvironments play critical regulatory roles in tumor growth. Although mouse cancer models have contributed to the understanding of human tumor biology, the effectiveness of mouse cancer models is limited by the inability of the models to accurately present humanized tumor microenvironments. Previously, a humanized breast cancer model in severe combined immunodeficiency mice was established, in which human breast cancer tissue was implanted subcutaneously, followed by injection of human breast cancer cells. It was demonstrated that breast cancer cells showed improved growth in the human mammary microenvironment compared with a conventional subcutaneous mouse model. In the present study, the novel mouse model and microarray technology was used to analyze changes in the expression of genes in breast cancer cells that are regulated by the human mammary microenvironment. Humanized breast and conventional subcutaneous mouse models were established, and orthotopic tumor cells were obtained from orthotopic tumor masses by primary culture. An expression microarray using Illumina HumanHT-12 v4 Expression BeadChip and database analyses were performed to investigate changes in gene expression between tumors from each microenvironment. A total of 94 genes were differentially expressed between the primary cells cultured from the humanized and conventional mouse models. Significant upregulation of genes that promote cell proliferation and metastasis or inhibit apoptosis, such as SH3-domain binding protein 5 (BTK-associated), sodium/chloride cotransporter 3 and periostin, osteoblast specific factor, and genes that promote angiogenesis, such as KIAA1618, was also noted. Other genes that restrain cell proliferation and accelerate cell apoptosis, including tripartite motif containing TRIM36 and NES1, were downregulated. The present results revealed differences in various aspects of tumor growth and metabolism between the two model groups and indicated the functional

  1. Imbalance between apoptosis and cell proliferation during early stages of mammary gland carcinogenesis in ACI rats.

    PubMed

    Kutanzi, Kristy R; Koturbash, Igor; Bronson, Roderick T; Pogribny, Igor P; Kovalchuk, Olga

    2010-12-10

    Estrogen and ionizing radiation are well-documented human breast carcinogens, yet the exact mechanisms of their deleterious effects on mammary gland remain to be discerned. Here we analyze the balance between cellular proliferation and apoptosis in the mammary glands of rats exposed to estrogen and X-ray radiation and the combined action of these carcinogenic agents. For the first time, we show that combined exposure to estrogen and radiation has a synergistic effect on cell proliferation in the mammary glands of ACI rats, as evidenced by a substantially greater magnitude of cell proliferation, especially after 12 and 18 weeks of treatment, when compared to mammary glands of rats exposed to estrogen or radiation alone. We also demonstrate that an imbalance between cell proliferation and apoptosis, rather than enhanced cell proliferation or apoptosis suppression alone, may be a driving force for carcinogenesis. Our studies further suggest that compromised functional activity of p53 may be one of the mechanisms responsible for the proliferation/apoptosis imbalance. In sum, the results of our study indicate that evaluation of the extent of cell proliferation and apoptosis before the onset of preneoplastic lesions may be a potential biomarker of breast cancer risk after exposure to breast carcinogens.

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

    PubMed

    Pang, Lisa Y; Cervantes-Arias, Alejandro; Else, Rod W; Argyle, David J

    2011-03-30

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

  3. 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

    SciTech Connect

    Burchiel, Scott W. . E-mail: SBurchiel@salud.unm.edu; Thompson, Todd A.; Lauer, Fredine T.; Oprea, Tudor I.

    2007-06-01

    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 as 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.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27558284

  5. Ligand-Independent Canonical Wnt Activity in Canine Mammary Tumor Cell Lines Associated with Aberrant LEF1 Expression

    PubMed Central

    van Wolferen, Monique E.; Rao, Nagesha A. S.; Grizelj, Juraj; Vince, Silvijo; Hellmen, Eva; Mol, Jan A.

    2014-01-01

    Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms. PMID:24887235

  6. Characterization of mammary cancer stem cells in the MMTV-PyMT mouse model.

    PubMed

    Ma, Jun; Lanza, Denise Grant; Guest, Ian; Uk-Lim, Chang; Glinskii, Anna; Glinsky, Gennadi; Sell, Stewart

    2012-12-01

    Breast cancer stem cells, the root of tumor growth, present challenges to investigate: Primary human breast cancer cells are difficult to establish in culture and inconsistently yield tumors after transplantation into immune-deficient recipient mice. Furthermore, there is limited characterization of mammary cancer stem cells in mice, the ideal model for the study of breast cancer. We herein describe a pre-clinical breast cancer stem cell model, based on the properties of cancer stem cells, derived from transgenic MMTV-PyMT mice. Using a defined set of cell surface markers to identify cancer stem cells by flow cytometry, at least four cell populations were recovered from primary mammary cancers. Only two of the four populations, one epithelial and one mesenchymal, were able to survive and proliferate in vitro. The epithelial population exhibited tumor initiation potential with as few as 10 cells injected into syngeneic immune-competent recipients. Tumors initiated from injected cell lines recapitulated the morphological and physiological components of the primary tumor. To highlight the stemness potential of the putative cancer stem cells, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) expression was knocked down via shRNA targeting Bmi-1. Without Bmi-1 expression, putative cancer stem cells could no longer initiate tumors, but tumor initiation was rescued with the introduction of a Bmi-1 overexpression vector in the Bmi-1 knockdown cells. In conclusion, our data show that primary mammary cancers from MMTV-PyMT mice contain putative cancer stem cells that survive in culture and can be used to create a model for study of mammary cancer stem cells.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. The chemokine receptor CCR7 promotes mammary tumorigenesis through amplification of stem-like cells.

    PubMed

    Boyle, S T; Ingman, W V; Poltavets, V; Faulkner, J W; Whitfield, R J; McColl, S R; Kochetkova, M

    2016-01-07

    The chemokine receptor CCR7 is widely implicated in breast cancer pathobiology. Although recent reports correlated high CCR7 levels with more advanced tumor grade and poor prognosis, limited in vivo data are available regarding its specific function in mammary gland neoplasia and the underlying mechanisms involved. To address these questions we generated a bigenic mouse model of breast cancer combined with CCR7 deletion, which revealed that CCR7 ablation results in a considerable delay in tumor onset as well as significantly reduced tumor burden. Importantly, CCR7 was found to exert its function by regulating mammary cancer stem-like cells in both murine and human tumors. In vivo experiments showed that loss of CCR7 activity either through deletion or pharmacological antagonism significantly decreased functional pools of stem-like cells in mouse primary mammary tumors, providing a mechanistic explanation for the tumor-promoting role of this chemokine receptor. These data characterize the oncogenic properties of CCR7 in mammary epithelial neoplasia and point to a new route for therapeutic intervention to target evasive cancer stem cells.

  9. Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

    PubMed

    Globerson-Levin, Anat; Waks, Tova; Eshhar, Zelig

    2014-05-01

    Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

  10. Human saliva as route of inter-human infection for mouse mammary tumor virus.

    PubMed

    Mazzanti, Chiara Maria; Lessi, Francesca; Armogida, Ivana; Zavaglia, Katia; Franceschi, Sara; Al Hamad, Mohammad; Roncella, Manuela; Ghilli, Matteo; Boldrini, Antonio; Aretini, Paolo; Fanelli, Giovanni; Marchetti, Ivo; Scatena, Cristian; Hochman, Jacob; Naccarato, Antonio Giuseppe; Bevilacqua, Generoso

    2015-07-30

    Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma.

  11. Human saliva as route of inter-human infection for mouse mammary tumor virus

    PubMed Central

    Armogida, Ivana; Zavaglia, Katia; Franceschi, Sara; Al Hamad, Mohammad; Roncella, Manuela; Ghilli, Matteo; Boldrini, Antonio; Aretini, Paolo; Fanelli, Giovanni; Marchetti, Ivo; Scatena, Cristian; Hochman, Jacob; Naccarato, Antonio Giuseppe; Bevilacqua, Generoso

    2015-01-01

    Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma. PMID:26214095

  12. 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

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

    SciTech Connect

    Chanson, L.; Brownfield, D.; Garbe, J. C.; Kuhn, I.; Stampfer, M. R.; Bissell, M. J.; LaBarge, M. A.

    2011-02-07

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

  14. MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.

    PubMed

    Aydoğdu, Eylem; Katchy, Anne; Tsouko, Efrosini; Lin, Chin-Yo; Haldosén, Lars-Arne; Helguero, Luisa; Williams, Cecilia

    2012-08-01

    MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

  15. 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.

  16. Dicer in Mammary Tumor Stem Cell Maintenance

    DTIC Science & Technology

    2007-03-01

    MicroRNAs ( miRNAs ) are a set of small RNAs produced by the RNAi machinery that play important functions in tissue organization and maintenance of cell... factor p53 is a tumor-suppressor gene that is deleted or mutated in many human cancers . To identify miRNAs that may be part of the p53 pathway, we...identity. Several miRNAs have been shown to collaborate with oncogenes in the progression of cancer , and in addition, miRNA expression profiling has

  17. Relative biological effectiveness of 25 and 10 kV X-rays for the induction of chromosomal aberrations in two human mammary epithelial cell lines.

    PubMed

    Beyreuther, Elke; Dörr, Wolfgang; Lehnert, Anna; Lessmann, Elisabeth; Pawelke, Jörg

    2009-08-01

    Administration of ionizing radiation for diagnostic purposes can be associated with a risk for the induction of tumors. Therefore, particularly with regard to general screening programs, e.g. with mammography, cost-benefit considerations must be discussed including risk estimation depending upon the radiation quality administered. The present study was initiated to investigate the in vitro X-ray energy dependence for the induction of chromosomal aberrations in the two mammary epithelial cell lines, 184A1 and MCF-12A. The induced excess fragments, dicentric chromosomes and centric rings were analyzed and the relative biological effectiveness (RBE) was determined for 10 and 25 kV X-rays relative to 200 kV X-rays. The assumed energy dependence with higher values for 10 kV X-rays was confirmed for the excess fragments, with RBE(M) values of 1.92 +/- 0.26 and 1.40 +/- 0.12 for 10 kV X-rays and 1.17 +/- 0.12 and 0.97 +/- 0.10 for 25 kV photons determined for cell lines 184A1 and MCF-12A, respectively. Meaningful results for the induction of dicentric chromosomes and centric rings were obtained only for higher doses with RBE values of 1.31 +/- 0.21 and 1.70 +/- 0.29 for 184A1 and 1.08 +/- 0.08 and 1.43 +/- 0.12 for MCF-12A irradiated with 25 and 10 kV X-rays, respectively.

  18. Role of the Stem Cell Niche in Hormone-Induced Tumorigenesis in Fetal Mouse Mammary Epithelium

    DTIC Science & Technology

    2005-08-01

    AD Award Number: W81XWH-04-1-0719 TITLE: Role of the Stem Cell Niche in Hormone-Induced Tumorigenesis in Fetal Mouse Mammary Epithelium PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Role of the Stem Cell Niche in Hormone-induced Tumorigenesis in Fetal Mouse 5b. GRANT NUMBER Mammary Epithelium...SUPPLEMENTARY NOTES 14. ABSTRACT SEE PAGE 4 15. SUBJECT TERMS Stem Cells , Stem Cell niche, Immunohistochemistry, mammary gland, breast cancer 16

  19. 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.

  20. Control of Differentiation of a Mammary Cell Line by Lipids

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

    A rat mammary cell line (LA7) undergoes spontaneous differentiation into domes due to production of specific inducers by the cells. Some of these inducers may be lipids, and we show that lipids regulate this differentiation as both inducers and inhibitors. One inhibitor is the tumor promoter tetradecanoyl-13 phorbol 12-acetate. The inducers are saturated fatty acids of two groups: butyric acid and acids with chain lengths from C13 to C16, especially myristic acid (C14). Other inducers are myristoyl and palmitoyl lysolecithins, myristic acid methyl ester, and two cationic detergents with a tetradecenyl chain. We propose that the lipids with a C14-C16 alkyl chain affect differentiation by recognizing specific receptors through their alkyl chains and that the effects obtained depend on the head groups. These lipids may be physiological regulators in the mammary gland.

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

    SciTech Connect

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

    2005-03-03

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

  2. Irradiation of juvenile, but not adult, mammary gland increases stem cell self-renewal and estrogen receptor negative tumors.

    PubMed

    Tang, Jonathan; Fernandez-Garcia, Ignacio; Vijayakumar, Sangeetha; Martinez-Ruis, Haydeliz; Illa-Bochaca, Irineu; Nguyen, David H; Mao, Jian-Hua; Costes, Sylvain V; Barcellos-Hoff, Mary Helen

    2014-03-01

    Children exposed to ionizing radiation have a substantially greater breast cancer risk than adults; the mechanism for this strong age dependence is not known. Here we show that pubertal murine mammary glands exposed to sparsely or densely ionizing radiation exhibit enrichment of mammary stem cell and Notch pathways, increased mammary repopulating activity indicative of more stem cells, and propensity to develop estrogen receptor (ER) negative tumors thought to arise from stem cells. We developed a mammary lineage agent-based model (ABM) to evaluate cell inactivation, self-renewal, or dedifferentiation via epithelial-mesenchymal transition (EMT) as mechanisms by which radiation could increase stem cells. ABM rejected cell inactivation and predicted increased self-renewal would only affect juveniles while dedifferentiation could act in both juveniles and adults. To further test self-renewal versus dedifferentiation, we used the MCF10A human mammary epithelial cell line, which recapitulates ductal morphogenesis in humanized fat pads, undergoes EMT in response to radiation and transforming growth factor β (TGFβ) and contains rare stem-like cells that are Let-7c negative or express both basal and luminal cytokeratins. ABM simulation of population dynamics of double cytokeratin cells supported increased self-renewal in irradiated MCF10A treated with TGFβ. Radiation-induced Notch concomitant with TGFβ was necessary for increased self-renewal of Let-7c negative MCF10A cells but not for EMT, indicating that these are independent processes. Consistent with these data, irradiating adult mice did not increase mammary repopulating activity or ER-negative tumors. These studies suggest that irradiation during puberty transiently increases stem cell self-renewal, which increases susceptibility to developing ER-negative breast cancer.

  3. Molecular Analysis of Motility in Metastatic Mammary Adenocarcinoma Cells

    DTIC Science & Technology

    1996-09-01

    Culture MTLn3 cells were clonally derived from a lung metastasis of the 13762NF rat mammary adenocarcinoma ( Neri et al., 1982) (kindly provided by Dr...MTLn3 cells were plated on collagen I coated MATTEK tissue culture dishes for 24 hours. Cells were plated at a density of 5000 cells/sq cm and...mM KOH; 4 mM MgC12 ; 10 mM EGTA pH 6.5 with 20 mM KOH; 5 1M phallacidin; 0.025 % saponin) was added to the culture well. After 15 seconds of extraction

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

    SciTech Connect

    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-{alpha} 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. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black

  5. Cellular quiescence in mammary stem cells and breast tumor stem cells: got testable hypotheses?

    PubMed

    Harmes, David C; DiRenzo, James

    2009-03-01

    Cellular quiescence is a state of reversible cell cycle arrest and has more recently been shown to be a blockade to differentiation and to correlate with resistance to cancer chemotherapeutics and other xenobiotics; features that are common to adult stem cells and possibly tumor stem cells. The biphasic kinetics of mammary regeneration, coupled to its cyclic endocrine control suggest that mammary stem cells most likely divide during a narrow window of the regenerative cycle and return to a state of quiescence. This would enable them to retain their proliferative capacity, resist differentiation signals and preserve their prolonged life span. There is accumulating evidence that mammary stem cells and other adult stem cells utilize quiescence for this purpose, however the degree to which tumor stem cells do so is largely unknown. The retained proliferative capacity of mammary stem cells likely enables them to accumulate and harbor mutations that lead to breast cancer initiation. However it is currently unclear if these causative lesions lead to defective or deranged quiescence in mammary stem cells. Evidence of such effects could potentially lead to the development of diagnostic systems that monitor mammary stem cell quiescence or activation. Such systems may be useful for the evaluation of patients who are at significant risk of breast cancer. Additionally quiescence has been postulated to contribute to therapeutic resistance and tumor recurrence. This review aims to evaluate what is known about the mechanisms governing cellular quiescence and the role of tumor stem cell quiescence in breast cancer recurrence.

  6. A role of ghrelin in canine mammary carcinoma cells proliferation, apoptosis and migration

    PubMed Central

    2012-01-01

    Background Ghrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay. Results The highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys3]-GHRP6) as well as RNA interference enhances early apoptosis. Conclusion The presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis. PMID:22999388

  7. Establishment and Characterization of a Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

    PubMed Central

    Anand, Vijay; Dogra, Nilambra; Singh, Surender; Kumar, Sudarshan N.; Jena, Manoj K.; Malakar, Dhruba; Dang, Ajay K.; Mishra, Bishnu P.; Mukhopadhyay, Tapas K.; Kaushik, Jai K.; Mohanty, Ashok K.

    2012-01-01

    Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions. PMID:22792341

  8. 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.

  9. Expression of the gene encoding growth hormone in the human mammary gland

    SciTech Connect

    Mol, J.A.; Misdorp, W.; Rijnberk, A.

    1995-10-01

    Progestins cause a syndrome of growth hormone (GH) excess and enhanced mammary tumorigenesis in the dog. This has been regarded as being specific for the dog. Recently we reported that progestin-induced GH excess originates from foci of hyperplastic ductular epithelium of the mammary gland in the dog. In the present report we demonstrate by reverse-transcriptase PCR and immunohistochemistry that a main factor involved in tissue growth, i.e. GH, is also expressed in normal and neoplastic human mammary glands. The gene expressed in the human mammary gland proved to be identical to the gene encoding GH in the pituitary gland. The role of progesterone in the GH expression of the human mammary gland needs, however, to be proven. It is hypothesized that this locally produced hGH may play a pathogenetic role in breast cancer. 21 refs., 2 figs., 1 tab.

  10. Generation of Induced Pluripotent Stem Cells from Bovine Epithelial Cells and Partial Redirection Toward a Mammary Phenotype In Vitro.

    PubMed

    Cravero, Diego; Martignani, Eugenio; Miretti, Silvia; Accornero, Paulo; Pauciullo, Alfredo; Sharma, Ruchi; Donadeu, Francesco Xavier; Baratta, Mario

    2015-06-01

    In contrast to adult stem cells, induced pluripotent stem cells (iPSCs) can be grown robustly in vitro and differentiated into virtually any tissue, thus providing an attractive alternative for biomedical applications. Although iPSC technology is already being used in human biomedicine, its potential in animal production has not been investigated. Herein, we investigated the potential application of iPSCs in dairy production by generating bovine iPSCs and establishing their ability to generate mammary epithelial tissue. iPSCs were derived by retrovirus-mediated expression of murine Oct4, Sox2, Klf4, and c-Myc in mammary epithelium and dermal fibroblasts. The resulting reprogrammed cells stained positive for alkaline phosphatase and showed renewed expression of pluripotency genes, including Lin28, Rex1, Oct4, Sox2, and Nanog. In addition, injection of epithelial- or fibroblast-derived reprogrammed cells into nonobese diabetic (NOD/NOD) mice resulted in the formation of teratomas containing differentiated derivatives of the three germ layers, including cartilage, membranous ossification, stratified squamous epithelial tissue, hair follicles, neural pinwheels, and different types of glandular tissue. Finally, mammary epithelium-derived iPSCs could be induced to differentiate back to a mammary phenotype characterized by epithelial cells expressing cytokeratin 14 (CK14), CK18, and smooth muscle actin (SMA) as a result of treatment with 10 nM progesterone. This study reports for the first time the generation of iPSCs from bovine epithelial cells and demonstrates the potential of using iPSCs technology for generating bovine mammary tissue in vitro.

  11. Is there a role for mammary stem cells in inflammatory breast carcinoma?: a review of evidence from cell line, animal model, and human tissue sample experiments.

    PubMed

    Van Laere, Steven; Limame, Ridha; Van Marck, Eric A; Vermeulen, Peter B; Dirix, Luc Y

    2010-06-01

    Stem cells are pluripotent cells, with a large replicative potential, which perform normal physiological functions such as tissue renewal and damage repair. However, because of their long lifespan and high replicative potential, stem cells are ideal targets to accumulate multiple mutations. Therefore, they can be regarded as being responsible for the initiation of tumor formation. In the past, numerous studies have shown that the presence of an elaborate stem cell compartment within a tumor is associated with aggressive tumor cell behavior, frequent formation of metastases, resistance to therapy, and poor patient survival. From this perspective, tumors from patients with inflammatory breast cancer (IBC), an aggressive breast cancer subtype with a dismal clinical course, are most likely to be associated with stem cell biology. To date, this hypothesis is corroborated by evidence resulting from in vitro and in vivo experiments. Both gene and microRNA expression profiles highlighted several stem cell-specific signal transduction pathways that are hyperactivated in IBC. Also, these stem cell-specific signal transduction pathways seem to converge in the activation of nuclear factor-kappa B, a molecular hallmark of IBC, and induction of epithelial-to-mesenchymal transition. Recently, the latter mechanism was identified as a prerequisite for the induction of stem cell characteristics in breast cancer cells.

  12. Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

    PubMed Central

    Barash, I; Nathan, M; Kari, R; Ilan, N; Shani, M; Hurwitz, D R

    1996-01-01

    Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals. PMID:8604300

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

    DTIC Science & Technology

    2013-02-01

    mammary epithelium impacts glandular development . We found ductal abnormali ties; however, the phenotype was not as severe as expected. Approximately...In previous reports we have clearly showed that cells w ith activated canonical Wnt signaling are present within the mammary epithelium starting at...Wnt1 transgenic cells. We generated a mouse line in which ~-catenin is conditionally deleted in the mammary epithelium of MMTV-Wnt1 transgenic

  14. 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

  15. Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of bis-(2-Chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

    PubMed Central

    Gruppi, Francesca; Hejazi, Leila; Christov, Plamen P.; Krishnamachari, Sesha; Turesky, Robert J.; Rizzo, Carmelo J.

    2015-01-01

    A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS3) method was established to characterize and measure five deoxyguanosine (dG) adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis-(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its crosslink (G-NM-G), the ring-opened formamidopyrimidine (FapyG) mono-adduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 107 DNA bases, when the equivalent of 5 μg DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G mono-adduct; the FapyG-NM-FapyG adduct was at the limit of detection. The NM-FapyG adducts formed in CT DNA at a level of ~20% that of the NM-G adduct. NM-FapyG has not been previously quanitified and the FapyG-NM-G and FapyG-NM-FapyG adducts have not be previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 107 bases), followed by G-NM-G (240 adducts per 107 bases) and NM-FapyG (180 adducts per 107 bases), and lastly the FapyG-NM-G cross-link adduct (6.0 adducts per 107 bases). These lesions are expected to contribute to the NM-mediated toxicity and

  16. 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.

  17. Cholera toxin enhances Na+ absorption across MCF10A human mammary epithelia

    PubMed Central

    Wang, Qian

    2013-01-01

    Cellular mechanisms to account for the low Na+ concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na+ channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na+ concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na+ transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body. PMID:24371040

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

    SciTech Connect

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

    2010-12-22

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

  19. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

    PubMed Central

    Bai, Lixia; Rohrschneider, Larry R.

    2010-01-01

    Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP+ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP+ cells can regenerate the mammary epithelial network. GFP+ mammary epithelial cells are p63+, CD24mod, CD49fhigh, and CD29high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP+ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells. PMID:20810647

  20. CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

    PubMed Central

    Spike, Benjamin T.; Kelber, Jonathan A.; Booker, Evan; Kalathur, Madhuri; Rodewald, Rose; Lipianskaya, Julia; La, Justin; He, Marielle; Wright, Tracy; Klemke, Richard; Wahl, Geoffrey M.; Gray, Peter C.

    2014-01-01

    Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers. PMID:24749068

  1. Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes.

    PubMed

    Prat, Aleix; Karginova, Olga; Parker, Joel S; Fan, Cheng; He, Xiaping; Bixby, Lisa; Harrell, J Chuck; Roman, Erick; Adamo, Barbara; Troester, Melissa; Perou, Charles M

    2013-11-01

    Five molecular subtypes (luminal A, luminal B, HER2-enriched, basal-like, and claudin-low) with clinical implications exist in breast cancer. Here, we evaluated the molecular and phenotypic relationships of (1) a large in vitro panel of human breast cancer cell lines (BCCLs), human mammary fibroblasts (HMFs), and human mammary epithelial cells (HMECs); (2) in vivo breast tumors; (3) normal breast cell subpopulations; (4) human embryonic stem cells (hESCs); and (5) bone marrow-derived mesenchymal stem cells (hMSC). First, by integrating genomic data of 337 breast tumor samples with 93 cell lines we were able to identify all the intrinsic tumor subtypes in the cell lines, except for luminal A. Secondly, we observed that the cell lines recapitulate the differentiation hierarchy detected in the normal mammary gland, with claudin-low BCCLs and HMFs cells showing a stromal phenotype, HMECs showing a mammary stem cell/bipotent progenitor phenotype, basal-like cells showing a luminal progenitor phenotype, and luminal B cell lines showing a mature luminal phenotype. Thirdly, we identified basal-like and highly migratory claudin-low subpopulations of cells within a subset of triple-negative BCCLs (SUM149PT, HCC1143, and HCC38). Interestingly, both subpopulations within SUM149PT were enriched for tumor-initiating cells, but the basal-like subpopulation grew tumors faster than the claudin-low subpopulation. Finally, claudin-low BCCLs resembled the phenotype of hMSCs, whereas hESCs cells showed an epithelial phenotype without basal or luminal differentiation. The results presented here help to improve our understanding of the wide range of breast cancer cell line models through the appropriate pairing of cell lines with relevant in vivo tumor and normal cell counterparts.

  2. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ) in normal mammary epithelial cells and breast tumors.

    PubMed

    Smart, Chanel E; Askarian Amiri, Marjan E; Wronski, Ania; Dinger, Marcel E; Crawford, Joanna; Ovchinnikov, Dmitry A; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T; Song, Sarah; Wiesner, Christiane; French, Juliet D; Dave, Richa K; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S; Lakhani, Sunil R; Brown, Melissa A; Kellie, Stuart

    2012-01-01

    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  3. CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

    PubMed Central

    Ramena, Grace; Yin, Yufang; Yu, Yang; Walia, Vijay; Elble, Randolph C.

    2016-01-01

    CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines. PMID:26930581

  4. Comparative aspects of mammary gland development and homeostasis.

    PubMed

    Capuco, Anthony V; Ellis, Steven E

    2013-01-01

    Mammary glands are crucial to the reproductive strategy of mammals, and the milk of domesticated ruminants serves as an important source of nutrients for the human population. The majority of mammary gland development occurs postnatally, and the mammary gland undergoes cyclical periods of growth, differentiation, lactation, and regression that are coordinated to provide nutrients for offspring or are driven by strategies to manage reproduction and milk production of domesticated species. Growth and maintenance of the mammary epithelium depends on the function of mammary stem cells and progenitor cells. In this review, we provide an overview of postnatal mammary gland development, cyclical phases of mammary gland regression (regression during lactation and between successive lactations), and mammary stem cells and progenitor cells. Where possible, these processes are related to animal production and compared across species, particularly bovine, porcine, murine, and human.

  5. The biology of zinc transport in mammary epithelial cells: implications for mammary gland development, lactation, and involution.

    PubMed

    McCormick, Nicholas H; Hennigar, Stephen R; Kiselyov, Kirill; Kelleher, Shannon L

    2014-03-01

    Zinc plays a critical role in a vast array of cellular functions including gene transcription, protein translation, cell proliferation, differentiation, bioenergetics, and programmed cell death. The mammary gland depends upon tight coordination of these processes during development and reproduction for optimal expansion, differentiation, and involution. For example, zinc is required for activation of matrix metalloproteinases, intracellular signaling cascades such as MAPK and PKC, and the activation of both mitochondrial-mediated apoptosis and lysosomal-mediated cell death. In addition to functional needs, during lactation the mammary gland must balance providing optimal zinc for cellular requirements with the need to secrete a substantial amount of zinc into milk to meet the requirements of the developing neonate. Finally, the mammary gland exhibits the most profound example of programmed cell death, which is driven by both apoptotic and lysosomal-mediated cell death. Two families of zinc-specific transporters regulate zinc delivery for these diverse functions. Members of the ZIP family of zinc transporters (ZIP1-14) import zinc into the cytoplasm from outside the cell or from subcellular organelles, while members of the ZnT family (ZnT1-10) export zinc from the cytoplasm. Recently, the ion channel transient receptor potential mucolipin 1 (TRPML1) has also been implicated in zinc transport. Herein, we review our current understanding of the molecular mechanisms through which mammary epithelial cells utilize zinc with a focus on the transport of zinc into discrete subcellular organelles for specific cellular functions during mammary gland development, lactation, and involution.

  6. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. A Synthetic Glycopeptide Vaccine for the Induction of a Monoclonal Antibody that Differentiates between Normal and Tumor Mammary Cells and Enables the Diagnosis of Human Pancreatic Cancer.

    PubMed

    Palitzsch, Björn; Gaidzik, Nikola; Stergiou, Natascha; Stahn, Sonja; Hartmann, Sebastian; Gerlitzki, Bastian; Teusch, Nicole; Flemming, Peer; Schmitt, Edgar; Kunz, Horst

    2016-02-18

    In studies within the realm of cancer immunotherapy, the synthesis of exactly specified tumor-associated glycopeptide antigens is shown to be a key strategy for obtaining a highly selective biological reagent, that is, a monoclonal antibody that completely differentiates between tumor and normal epithelial cells and specifically marks the tumor cells in pancreas tumors. Mucin MUC1, which is overexpressed in many prevalent cancers, was identified as a promising target for this strategy. Tumor-associated MUC1 differs significantly from that expressed by normal cells, in particular by altered glycosylation. Structurally defined tumor-associated MUC1 cannot be isolated from tumor cells. We synthesized MUC1-glycopeptide vaccines and analyzed their structure-activity relationships in immunizations; a monoclonal antibody that specifically distinguishes between human normal and tumor epithelial cells was thus generated.

  8. The dog as a natural animal model for study of the mammary myoepithelial basal cell lineage and its role in mammary carcinogenesis.

    PubMed

    Rasotto, R; Goldschmidt, M H; Castagnaro, M; Carnier, P; Caliari, D; Zappulli, V

    2014-01-01

    Basal-like tumours constitute 2-18% of all human breast cancers (HBCs). These tumours have a basal myoepithelial phenotype and it has been hypothesized that they originate from either myoepithelial cells or mammary progenitor cells. They are heterogeneous in morphology, clinical presentation, outcome and response to therapy. Canine mammary carcinomas (CMCs) have epidemiological and biological similarities to HBCs, are frequently biphasic and are composed of two distinct neoplastic populations (epithelial and myoepithelial). The present study evaluates the potential of CMCs as a natural model for basal-like HBCs. Single and double immunohistochemistry was performed on serial sections of 10 normal canine mammary glands and 65 CMCs to evaluate expression of cytokeratin (CK) 8/18, CK5, CK14, α-smooth muscle actin (SMA), calponin (CALP), p63 and vimentin (VIM). The tumours were also evaluated for Ki67 and human epidermal growth factor receptor (HER)-2 expression. A hierarchical model of cell differentiation was established, similar to that for the human breast. We hypothesized that progenitor cells (CK5(+), CK14(+), p63(+) and VIM(+)) differentiate into terminally-differentiated luminal glandular (CK8/18(+)) and myoepithelial (CALP(+), SMA(+) and VIM(+)) cells via intermediary luminal glandular cells (CK5(+), CK14(+) and CK8/CK18(+)) and intermediary myoepithelial cells (CK5(+), CK14(+), p63(+), SMA(+), CALP(+) and VIM(+)). Neoplastic myoepithelial cells in canine complex carcinomas had labelling similar to that of terminally-differentiated myoepithelial cells, while those of carcinomas-and-malignant myoepitheliomas with a more aggressive biological behaviour (i.e. higher frequency of vascular/lymph node invasion and visceral metastases and higher risk of tumour-related death) were comparable with intermediary myoepithelial cells and had significantly higher Ki67 expression. The majority of CMCs examined were negative for expression of HER-2. The biphasic appearance of

  9. EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells

    SciTech Connect

    Rodriguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G.; Lauer, Fredine T.; Burchiel, Scott W.

    2009-03-15

    Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-{gamma}1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 {mu}M), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-{gamma}1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-{gamma}1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-{gamma}1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

  10. Overexpression of LMO4 induces mammary hyperplasia, promotes cell invasion, and is a predictor of poor outcome in breast cancer

    PubMed Central

    Sum, Eleanor Y. M.; Segara, Davendra; Duscio, Belinda; Bath, Mary L.; Field, Andrew S.; Sutherland, Robert L.; Lindeman, Geoffrey J.; Visvader, Jane E.

    2005-01-01

    The zinc finger protein LMO4 is overexpressed in a high proportion of breast carcinomas. Here, we report that overexpression of a mouse mammary tumor virus (MMTV)-Lmo4 transgene in the mouse mammary gland elicits hyperplasia and mammary intraepithelial neoplasia or adenosquamous carcinoma in two transgenic strains with a tumor latency of 13–18 months. To investigate cellular processes controlled by LMO4 and those that may be deregulated during oncogenesis, we used RNA interference. Down-regulation of LMO4 expression reduced proliferation of human breast cancer cells and increased differentiation of mouse mammary epithelial cells. Furthermore, small-interfering-RNA-transfected breast cancer cells (MDA-MB-231) had a reduced capacity to migrate and invade an extracellular matrix. Conversely, overexpression of LMO4 in noninvasive, immortalized human MCF10A cells promoted cell motility and invasion. Significantly, in a cohort of 159 primary breast cancers, high nuclear levels of LMO4 were an independent predictor of death from breast cancer. Together, these findings suggest that deregulation of LMO4 in breast epithelium contributes directly to breast neoplasia by altering the rate of cellular proliferation and promoting cell invasion. PMID:15897450

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Diet Does Not Affect Putative Mammary Epithelial Stem Cells in Pre-weaned Holstein Heifers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Overfeeding prepubertal heifers can impair mammary epithelial growth and development, processes that depend on stem cells. In this study we evaluated effects of diet composition on putative bovine mammary epithelial stem cell populations using a 5-bromo-2-deoxyrudine (BrdU; a thymidine analog) label...

  13. APOBEC3 proteins expressed in mammary epithelial cells are packaged into retroviruses and can restrict transmission of milk-borne virions.

    PubMed

    Okeoma, Chioma M; Huegel, Alyssa L; Lingappa, Jaisri; Feldman, Michael D; Ross, Susan R

    2010-12-16

    Viruses, including retroviruses like human immunodeficiency virus (HIV) and mouse mammary tumor virus (MMTV), are transmitted from mother to infants through milk. Lymphoid cells and antibodies are thought to provide mammary gland and milk-borne immunity. In contrast, little is known about the role of mammary epithelial cells (MECs). The APOBEC3 family of retroviral restriction factors is highly expressed in macrophages and lymphoid and dendritic cells. We now show that APOBEC3 proteins are also expressed in mouse and human MECs. Lymphoid cell-expressed APOBEC3 restricts in vivo spread of MMTV to lymphoid and mammary tissue. In contrast, mammary gland-expressed APOBEC3 is packaged into MMTV virions and decreases the infectivity of milk-borne viruses. Moreover, APOBEC3G and other APOBEC3 genes are expressed in human mammary cells and have the potential to restrict viruses produced in this cell type. These data point to a role for APOBEC3 proteins in limiting infectivity of milk-transmitted viruses.

  14. Weight gain increases human aromatase expression in mammary gland.

    PubMed

    Chen, Dong; Zhao, Hong; Coon, John S; Ono, Masanori; Pearson, Elizabeth K; Bulun, Serdar E

    2012-05-15

    Adulthood weight gain predicts estrogen receptor-positive breast cancer. Because local estrogen excess in the breast likely contributes to cancer development, and aromatase is the key enzyme in estrogen biosynthesis, we investigated the role of local aromatase expression in weight gain-associated breast cancer risk in a humanized aromatase (Arom(hum)) mouse model containing the coding region and the 5'-regulatory region of the human aromatase gene. Compared with littermates on normal chow, female Arom(hum) mice on a high fat diet gained more weight, and had a larger mammary gland mass with elevated total human aromatase mRNA levels via promoters I.4 and II associated with increased levels of their regulators TNFα and C/EBPβ. There was no difference in total human aromatase mRNA levels in gonadal white adipose tissue. Our data suggest that diet-induced weight gain preferentially stimulates local aromatase expression in the breast, which may lead to local estrogen excess and breast cancer risk.

  15. Anatomy of the human mammary gland: Current status of knowledge.

    PubMed

    Hassiotou, Foteini; Geddes, Donna

    2013-01-01

    Mammary glands are unique to mammals, with the specific function of synthesizing, secreting, and delivering milk to the newborn. Given this function, it is only during a pregnancy/lactation cycle that the gland reaches a mature developmental state via hormonal influences at the cellular level that effect drastic modifications in the micro- and macro-anatomy of the gland, resulting in remodeling of the gland into a milk-secretory organ. Pubertal and post-pubertal development of the breast in females aids in preparing it to assume a functional state during pregnancy and lactation. Remarkably, this organ has the capacity to regress to a resting state upon cessation of lactation, and then undergo the same cycle of expansion and regression again in subsequent pregnancies during reproductive life. This plasticity suggests tight hormonal regulation, which is paramount for the normal function of the gland. This review presents the current status of knowledge of the normal macro- and micro-anatomy of the human mammary gland and the distinct changes it undergoes during the key developmental stages that characterize it, from embryonic life through to post-menopausal age. In addition, it discusses recent advances in our understanding of the normal function of the breast during lactation, with special reference to breastmilk, its composition, and how it can be utilized as a tool to advance knowledge on normal and aberrant breast development and function. Finally, anatomical and molecular traits associated with aberrant expansion of the breast are discussed to set the basis for future comparisons that may illuminate the origin of breast cancer.

  16. Regulation of Mammary Progenitor Cells by p53 and Parity

    DTIC Science & Technology

    2011-01-01

    2003). 12. Ibarra , I., Erlich, Y., Muthuswamy, S. K., Sachidanandam, R. & Hannon, G. J. A role for microRNAs in maintenance of mouse mammary...supplemented with 1mM EDTA, 25mM HEPES, 1%FBS and 100u/ml Pen/Strep. The FACS data were collected using LSRII (Becton Dickinson, San Jose CA). A total...epithelial stem/progenitor cells. BREAST CANCER RES. 2009;11(2):R20. 28. Ibarra I, Erlich Y, Muthuswamy SK et al. A role for microRNAs in maintenance

  17. 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

  18. 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

  19. Progenitor Cell Fate Decisions in Mammary Tumorigenesis

    DTIC Science & Technology

    2013-03-01

    luminal progenitors contributing to transformation of ER- luminal and basal cells and development of treatment resistant breast cancer . We previously...proliferate and metastasize. Decreased DNA damage repair or altered epigenetic marks can dramatically affect the cellular composition of these tumors

  20. 8-Prenylnaringenin, the phytoestrogen in hops and beer, upregulates the function of the E-cadherin/catenin complex in human mammary carcinoma cells.

    PubMed

    Rong, H; Boterberg, T; Maubach, J; Stove, C; Depypere, H; Van Slambrouck, S; Serreyn, R; De Keukeleire, D; Mareel, M; Bracke, M

    2001-09-01

    The E-cadherin/catenin complex is a powerful invasion suppressor in epithelial cells. It is expressed in the human MCF-7 breast cancer cell line family, but functionally defective in the invasive MCF-7/6 variant. Previous experiments have shown that IGF-I, tamoxifen, retinoic acid and tangeretin are able to upregulate the function of this complex in MCF-7/6 cells. We investigated the effect of 8-prenylnaringenin (8-PN), the phytoestrogen present in hops and beer, on aggregation, growth and invasion in MCF-7/6 cells. 8-PN was found to stimulate E-cadherin-dependent aggregation and growth of MCF-7/6 cells in suspension. These effects could be inhibited by the pure anti-estrogen ICI 182,780. 8-PN did not affect invasion of MCF-7/6 cells in the chick heart assay in vitro. In all these aspects 8-PN mimics the effects of 17beta-estradiol on MCF-7/6 cells.

  1. Dicer in Mammary Tumor Stem Cell Maintenance

    DTIC Science & Technology

    2008-03-01

    SUPPLEMENTARY NOTES 14. ABSTRACT To date, most cancer research has focused on alterations in the sequence, gene structure, copy number and expression of...To address the role of miR-34in cancer formation and maintenance, we generated cell lines over express miR-34. We have demonstrated that ectopic...mediators of p53 tumor suppressor network, which plays an important role in many cancer types, including breast cancer . 15. SUBJECT TERMS Dicer

  2. Dicer in Mammary Tumor Stem Cell Maintenance

    DTIC Science & Technology

    2006-03-01

    a gene silencing pathway with roles in mRNA stability, translational control, chromatin organization and genome regulation. MicroRNAs ( miRNAs ) are a... miRNAs have been shown to collaborate with oncogenes in the progression of cancer , and in addition, miRNA expression profiling has revealed widespread... miRNA misregulation in cancer . To address the role of miRNAs in the onset and maintenance of breast cancer , we have created embryonic stem (ES) cells

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

    SciTech Connect

    Talhouk, R.S.

    1988-01-01

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

  4. MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

    PubMed Central

    Haikala, Heidi M.; Klefström, Juha; Eilers, Martin; Wiese, Katrin E.

    2016-01-01

    ABSTRACT Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue. PMID:26873145

  5. Remodeling of endogenous mammary epithelium by breast cancer stem cells.

    PubMed

    Parashurama, Natesh; Lobo, Neethan A; Ito, Ken; Mosley, Adriane R; Habte, Frezghi G; Zabala, Maider; Smith, Bryan R; Lam, Jessica; Weissman, Irving L; Clarke, Michael F; Gambhir, Sanjiv S

    2012-10-01

    Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.

  6. Bimodal Analysis of Mammary Epithelial Cell Migration in Two Dimensions

    PubMed Central

    Potdar, Alka A.; Lu, Jenny; Jeon, Junhwan; Weaver, Alissa M.; Cummings, Peter T.

    2013-01-01

    Cell migration paths of mammary epithelial cells (expressing different versions of the promigratory tyrosine kinase receptor Her2/Neu) were analyzed within a bimodal framework that is a generalization of the run-and-tumble description applicable to bacterial migration. The mammalian cell trajectories were segregated into two types of alternating modes, namely, the “directional-mode” (mode I, the more persistent mode, analogous to the bacterial run phase) and the “re-orientation-mode” (mode II, the less persistent mode, analogous to the bacterial tumble phase). Higher resolution (more pixel information, relative to cell size) and smaller sampling intervals (time between images) were found to give a better estimate of the deduced single cell dynamics (such as directional-mode time and turn angle distribution) of the various cell types from the bimodal analysis. The bimodal analysis tool permits the deduction of short-time dynamics of cell motion such as the turn angle distributions and turn frequencies during the course of cell migration compared to standard methods of cell migration analysis. We find that the two-hour mammalian cell tracking data do not fall into the diffusive regime implying that the often-used random motility expressions for mammalian cell motion (based on assuming diffusive motion) are invalid over the time steps (fraction of minute) typically used in modeling mammalian cell migration. PMID:18982450

  7. Precursors of hexoneogenesis within the human mammary gland.

    PubMed

    Mohammad, Mahmoud A; Maningat, Patricia; Sunehag, Agneta L; Haymond, Morey W

    2015-04-15

    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 breastfeeding women were studied following a 24-h fast on two occasions separated by 1-3 wk. Five women were infused with [U-¹³C]lactate or [1,2-¹³C₂]glutamine and five women with [U-¹³C]glycerol or [1,2-¹³C₂]acetate. Enrichments of ¹³C in plasma and milk substrates were analyzed using GC-MS. Infusion of labeled lactate, glycerol, glutamine, and acetate resulted in plasma glucose being 22.0±3.7, 11.2±1.0, 2.5±0.5, and 1.3±0.2% labeled, respectively. Lactate, glutamine, or acetate did not contribute to milk glucose or galactose (0-2%). In milk, ¹³C-free glycerol enrichment was one-fourth that in plasma but free glycerol concentration in milk was fourfold higher than in plasma. Using [U-¹³C]glycerol and by accounting for tracer dilution, glycerol alone contributed to 10±2 and 69±11% of the hexoneogenesis of milk glucose and galactose, respectively. During [U-¹³C]glycerol infusion, the ratio of M₃ enrichment on 4-6 carbons/M₃ on 1-3 carbons of galactose was higher (P<0.05, 1.22±0.05) than those of glucose in plasma (1.05±0.03) and milk (1.07±0.02). Reanalysis of samples from a previous study involving [U-¹³C]glucose infusion alone suggested labeling a portion of galactose consistent with pentose phosphate pathway (PPP) activity. We conclude that, although lactate contributed significantly to gluconeogenesis, glycerol alone provides the vast majority of substrate for hexoneogenesis. The relative contribution of the PPP vs. the reversal Embden-Meyerhof pathway to hexoneogenesis within the human mammary gland remains to be determined.

  8. Associated expressions of FGFR-2 and FGFR-3: from mouse mammary gland physiology to human breast cancer.

    PubMed

    Cerliani, Juan P; Vanzulli, Silvia I; Piñero, Cecilia Pérez; Bottino, María C; Sahores, Ana; Nuñez, Myriam; Varchetta, Romina; Martins, Rubén; Zeitlin, Eduardo; Hewitt, Stephen M; Molinolo, Alfredo A; Lanari, Claudia; Lamb, Caroline A

    2012-06-01

    Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors which have been implicated in breast cancer. The aim of this study was to evaluate FGFR-1, -2, -3, and -4 protein expressions in normal murine mammary gland development, and in murine and human breast carcinomas. Using immunohistochemistry and Western blot, we report a hormonal regulation of FGFR during postnatal mammary gland development. Progestin treatment of adult virgin mammary glands resulted in changes in localization of FGFR-3 from the cytoplasm to the nucleus, while treatment with 17-β-estradiol induced changes in the expressions and/or localizations of FGFR-2 and -3. In murine mammary carcinomas showing different degrees of hormone dependence, we found progestin-induced increased expressions, mainly of FGFR-2 and -3. These receptors were constitutively activated in hormone-independent variants. We studied three luminal human breast cancer cell lines growing as xenografts, which particularly expressed FGFR-2 and -3, suggesting a correlation between hormonal status and FGFR expression. Most importantly, in breast cancer samples from 58 patients, we found a strong association (P < 0.01; Spearman correlation) between FGFR-2 and -3 expressions and a weaker correlation of each receptor with estrogen receptor expression. FGFR-4 correlated with c-erbB2 over expression. We conclude that FGFR-2 and -3 may be mechanistically linked and can be potential targets for treatment of estrogen receptor-positive breast cancer patients.

  9. CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin.

    PubMed

    John, Kaarthik; Divi, Rao L; Keshava, Channa; Orozco, Christine C; Schockley, Marie E; Richardson, Diana L; Poirier, Miriam C; Nath, Joginder; Weston, Ainsley

    2010-06-28

    Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4muM) in the absence or presence of chlorophyllin (5muM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p<0.005) and CYP1B1 expression in 17/20 NHMEC strains (p<0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p<0.005 for CYP1A1 and CYP1B1 expression and p<0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.

  10. p16 loss rescues functional decline of Brca1-deficient mammary stem cells.

    PubMed

    Scott, Alexandria; Bai, Feng; Chan, Ho Lam; Liu, Shiqin; Slingerland, Joyce M; Robbins, David J; Capobianco, Anthony J; Pei, Xin-Hai

    2017-02-22

    Recent evidence indicates that the accumulation of endogenous DNA damage can induce senescence and limit the function of adult stem cells. It remains elusive whether deficiency in DNA damage repair is associated with the functional alteration of mammary stem cells. In this article, we reported that senescence was induced in mammary epithelial cells during aging along with increased expression of p16Ink4a (p16), an inhibitor of CDK4 and CKD6. Loss of p16 abrogated the age-induced senescence in mammary epithelial cells and significantly increased mammary stem cell function. We showed that loss of Brca1, a tumor suppressor that functions in DNA damage repair, in the mammary epithelium induced senescence with induction of p16 and a decline of stem cell function, which was rescued by p16 loss. These data not only answer the question as to whether deficiency in DNA damage repair is associated with the functional decline of mammary stem cells, but also identify the role of p16 in suppressing Brca1-deficient mammary stem cell function.

  11. 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

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

    PubMed Central

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

    2014-01-01

    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. PMID:24903350

  13. 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.

  14. Difference between mammary epithelial cells from mature virgin and primiparous mice.

    PubMed

    Vonderhaar, B K; Smith, G H; Pauley, R J; Rosen, J M; Topper, Y J

    1978-11-01

    Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

  15. Mathematical analysis of mammary ducts in lactating human breast.

    PubMed

    Mortazavi, S Negin; Geddes, Donna; Hassiotou, Foteini; Hassanipour, Fatemeh

    2014-01-01

    This work studies a simple model for milk transport through lactating human breast ducts, and describes mathematically the mass transfer from alveolar sacs through the mammary ducts to the nipple. In this model both the phenomena of diffusion in the sacs and conventional flow in ducts have been considered. The ensuing analysis reveals that there is an optimal range of bifurcation numbers leading to the easiest milk flow based on the minimum flow resistance. This model formulates certain difficult-to-measure values like diameter of the alveolar sacs, and the total length of the milk path as a function of easy-to-measure properties such as milk fluid properties and macroscopic measurements of the breast. Alveolar dimensions from breast tissues of six lactating women are measured and reported in this paper. The theoretically calculated alveoli diameters for optimum milk flow (as a function of bifurcation numbers) show excellent match with our biological data on alveolar dimensions. Also, the mathematical model indicates that for minimum milk flow resistance the glandular tissue must be within a short distance from the base of the nipple, an observation that matches well with the latest anatomical and physiological research.

  16. Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters

    PubMed Central

    Naba, Alexandra; Clauser, Karl R; Lamar, John M; Carr, Steven A; Hynes, Richard O

    2014-01-01

    The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER−/PR−breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools. DOI: http://dx.doi.org/10.7554/eLife.01308.001 PMID:24618895

  17. Mammary epithelial cell: Influence of extracellular matrix composition and organization during development and tumorigenesis

    PubMed Central

    Kass, Laura; Erler, Janine T.; Dembo, Micah; Weaver, Valerie M.

    2009-01-01

    Stromal–epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal–epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation. PMID:17719831

  18. Establishment and characterization of a new cell line of canine inflammatory mammary cancer: IPC-366.

    PubMed

    Caceres, Sara; Peña, Laura; de Andres, Paloma J; Illera, Maria J; Lopez, Mirtha S; Woodward, Wendy A; Reuben, James M; Illera, Juan C

    2015-01-01

    Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative

  19. Establishment and Characterization of a New Cell Line of Canine Inflammatory Mammary Cancer: IPC-366

    PubMed Central

    Caceres, Sara; Peña, Laura; de Andres, Paloma J.; Illera, Maria J.; Lopez, Mirtha S.; Woodward, Wendy A.; Reuben, James M.; Illera, Juan C.

    2015-01-01

    Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative

  20. Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization

    SciTech Connect

    Cerosaletti, K.M.; Shapero, M.H.; Fournier, R.E.K.

    1995-01-01

    We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region. 47 refs., 6 figs.

  1. Mammary cancer and epithelial stem cells: a problem or a solution?

    PubMed

    Smith, Gilbert H

    2002-01-01

    The existing paradigms for stem cells in adult tissues include the integument, the alimentary canal, the lung, the liver, skeletal muscle and bone marrow. The mammary gland, by contrast, is the 'new kid on the block'. What little is known about stem cells in the mammary gland indicates that they possess a prodigious capacity for self-renewal. More importantly, in rodents, they persist with undiminished reproductive vigor throughout the organism's lifetime without regard to age or reproductive history. Do these stem cells represent primary targets for mammary neoplasia? If so, what are the implications for prevention/therapy?

  2. Characterization of HOX gene expression in canine mammary tumour cell lines from spontaneous tumours.

    PubMed

    DeInnocentes, P; Perry, A L; Graff, E C; Lutful Kabir, F M; Curtis Bird, R

    2015-09-01

    Spatial/temporal controls of development are regulated by the homeotic (HOX) gene complex and require integration with oncogenes and tumour suppressors regulating cell cycle exit. Spontaneously derived neoplastic canine mammary carcinoma cell models were investigated to determine if HOX expression profiles were associated with neoplasia as HOX genes promote neoplastic potential in human cancers. Comparative assessment of human and canine breast cancer expression profiles revealed remarkable similarity for all four paralogous HOX gene clusters and several unlinked HOX genes. Five canine HOX genes were overexpressed with expression profiles consistent with oncogene-like character (HOXA1, HOXA13, HOXD4, HOXD9 and SIX1) and three HOX genes with underexpressed profiles (HOXA11, HOXC8 and HOXC9) were also identified as was an apparent nonsense mutation in HOXC6. This data, as well as a comparative analysis of similar data from human breast cancers suggested expression of selected HOX genes in canine mammary carcinoma could be contributing to the neoplastic phenotype.

  3. A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer.

    PubMed

    von Eyss, Björn; Jaenicke, Laura A; Kortlever, Roderik M; Royla, Nadine; Wiese, Katrin E; Letschert, Sebastian; McDuffus, Leigh-Anne; Sauer, Markus; Rosenwald, Andreas; Evan, Gerard I; Kempa, Stefan; Eilers, Martin

    2015-12-14

    In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

  4. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

    SciTech Connect

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua . E-mail: jiaobh@uninet.com.cn; Cheng Guoxiang . E-mail: Chenggx@cngenon.com

    2005-07-22

    Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo{sup r}), replaced the {alpha}-lactalbumin gene in a 210 kb human {alpha}-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.

  5. Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.

    PubMed

    Lipnik, Karoline; Naschberger, Elisabeth; Gonin-Laurent, Nathalie; Kodajova, Petra; Petznek, Helga; Rungaldier, Stefanie; Astigiano, Simonetta; Ferrini, Silvano; Stürzl, Michael; Hohenadl, Christine

    2010-01-01

    Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

  6. Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine.

    PubMed

    Martignani, Eugenio; Cravero, Diego; Miretti, Silvia; Accornero, Paolo; Baratta, Mario

    2015-11-01

    Adult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors. We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells. When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted. We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology.

  7. Identification of Stem Cells in a Novel Human Mammary Epithelial Culture (HMEC) System that Reproducibly Demonstrates Ductal Organotypic Architecture in 3 Weeks

    DTIC Science & Technology

    2005-10-01

    maintaining HME stem cells in vitro. Clinically this study on stem cells will allow many laboratories to test the long held hypothesis that some...for CD24, a very marked difference between these cells and the BRL cell lines tested . In addition a very different profile for CD90 and CD81 was...different conditions were tested for 2 of these cell lines but cloning is achievable for all. 17 Fig. 12. Single cell pick up was physically

  8. Identification of Stem Cells in a Novel Human Mammary Epithelial Culture (HMEC) System that Reproducibly Demonstrates Ductal Organotypic Architecture in 3 Weeks

    DTIC Science & Technology

    2006-10-01

    maintaining HME stem cells in vitro. Clinically this study on stem cells will allow many laboratories to test the long held hypothesis that some...for CD24, a very marked difference between these cells and the BRL cell lines tested . In addition a very different profile for CD90 and CD81 was...different conditions were tested for 2 of these cell lines but cloning is achievable for all. 17 Fig. 12. Single cell pick up was physically

  9. Short SULF1/SULF2 splice variants predominate in mammary tumours with a potential to facilitate receptor tyrosine kinase-mediated cell signalling.

    PubMed

    Gill, Roop Ms; Mehra, Vedika; Milford, Emma; Dhoot, Gurtej K

    2016-10-01

    The relative roles of SULF1 and SULF2 enzymes in tumour growth are controversial, but short SULF1/SULF2 splice variants predominate in human mammary tumours despite their non-detectable levels in normal mammary tissue. Compared with the normal, the level of receptor tyrosine kinase (RTK) activity was markedly increased in triple-positive mammary tumours during later stages of tumour progression showing increased p-EGFR, p-FGFR1 and p-cMet activity in triple-positive but not in triple-negative tumours. The abundance of catalytically inactive short SULF1/SULF2 variants permits high levels of HS sulphation and thus growth driving RTK cell signalling in primary mammary tumours. Also observed in this study, however, was increased N-sulphation detected by antibody 10E4 indicating that not only 6-O sulphation but also N-sulphation may contribute to increased RTK cell signalling in mammary tumours. The levels of such increases in not only SULF1/SULF2 but also in pEGFR, pFGFR1, p-cMet and Smad1/5/8 signalling were further enhanced following lymph node metastasis. The over-expression of Sulf1 and Sulf2 variants in mammary tumour-derived MDA-MB231 and MCF7 cell lines by transfection further confirms Sulf1-/Sulf2-mediated differential modulation of growth. The short variants of both Sulf1 and Sulf2 promoted FGF2-induced MDA-MB231 and MCF7 in vitro growth while full-length Sulf1 inhibited growth supporting in vivo mammary tumour cell signalling patterns of growth. Since a number of mammary tumours become drug resistant to hormonal therapy, Sulf1/Sulf2 inhibition could be an alternative therapeutic approach to target such tumours by down-regulating RTK-mediated cell signalling.

  10. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    PubMed

    Campbell, Jonathan J; Davidenko, Natalia; Caffarel, Maria M; Cameron, Ruth E; Watson, Christine J

    2011-01-01

    Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  11. Platelet factors induce chemotactic migration of murine mammary adenocarcinoma cells with different metastatic capabilities.

    PubMed Central

    Sarach, M. A.; Rovasio, R. A.; Eynard, A. R.

    1993-01-01

    The chemotactic response of neoplastic cells (NC) induced by soluble platelet factors was investigated. NC suspensions isolated from murine mammary gland adenocarcinomas having different metastatic capabilities were incubated in Boyden's chambers and challenged with (1) 'Early Platelet Factors' (EP), obtained from the soluble fraction of recently collagen-activated human platelets, and (2) 'Late Platelet Factors' (LP), isolated after 24 hours incubation of the platelet aggregates. Chemotaxis was expressed as the distance travelled by NC through nitrocellulose filters. NC isolated from M3, the tumour line having the stronger metastatic potential, showed a significant chemotactic response towards LP factors, whereas NC from the M2 line exhibiting the lower metastatic behaviour, showed a chemotactic response towards EP factors. Both tumour cell lines lacked motion capability towards the well known chemoattractant peptide N-f-Met-Leu-Phe-Phe as well as to serum, plasma, collagen type I or culture medium. The different chemotactic response of both tumour lines when they were challenged by concentration gradients of factors released by early or late collagen-activated human platelets, confirm a relationship between platelet activity and metastatic capabilities and suggests that platelet chemoattractants might play a role in the metastatic dissemination of these mammary gland adenocarcinomas. Images Figure 1 PMID:8217786

  12. Histopathological findings in a highly invasive mouse mammary carcinoma transfected with human tissue inhibitor of metalloproteinases-1.

    PubMed

    Alonso, D F; Skilton, G; De Lorenzo, M S; Scursoni, A M; Yoshiji, H; Gomez, D E

    1998-01-01

    We have transfected a full-lenght cDNA-encoding human tissue inhibitor of metalloproteinases-1 (TIMP-1) by lipofection in highly invasive F3II mouse mammary sarcomatoid carcinoma cells. In vitro, overexpression of TIMP-1 was associated with abrogation of metalloproteinase activity, extended doubling time, and a more flattened, epithelioid polyhedric morphology. Female Balb/c mice inoculated subcutaneously with TIMP-1 transfectant exhibited a prolonged tumor latency and tumor burden was significantly lower in early stages of tumor growth. Control F3II cells grew by invading the muscular and adipose layers of the subcutis, dermis, and dermal papillae. On the contrary, mammary carcinoma cells transfected with TIMP-1 grew without signs of active invasion of dermis. Tumors also revealed a decreased amount of necrosis and host inflammatory cell infiltrates. However, histological analysis did not demonstrate any change in vascular density. Animals bearing F3II tumors overexpressing TIMP-1 showed a significant reduction in the size of metastatic lung nodules. These data suggested that TIMP-1 overexpression may reduce local invasion and delay the progression of the metastatic disease in the present mammary tumor model.

  13. Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells.

    PubMed

    Pecorini, Chiara; Delpal, Serge; Truchet, Sandrine; Le Provost, Fabienne; Baldi, Antonella; Ollivier-Bousquet, Michèle

    2009-11-01

    Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.

  14. PARP Inhibitors Synergize With Loss of Checkpoint Control to Kill Mammary Carcinoma Cells

    DTIC Science & Technology

    2011-06-01

    term and long-term colony assays, inhibition of PARP1 CHK1 function enhanced the toxic effects of exposure to ionizing radiation (Fig. 3G). In Figs...3. Does PARP1inhibitor + Chk1 inhibitor treatment radiosensitize mammary tumor cells in vitro? Task 3 has been completed ( 1 ). We have extended...combination effect as tumors growing in the mammary fat pad. On-going studies are examining the effect of the drugs on MCF7 cells . Task 5

  15. A model of spontaneous mouse mammary tumor for human estrogen receptor- and progesterone receptor-negative breast cancer

    PubMed Central

    ZHENG, LIXIANG; ZHOU, BUGAO; MENG, XIANMING; ZHU, WEIFENG; ZUO, AIREN; WANG, XIAOMIN; JIANG, RUNDE; YU, SHIPING

    2014-01-01

    Breast cancer (BC) is the most frequently malignancy in women. Therefore, establishment of an animal model for the development of preventative measures and effective treatment for tumors is required. A novel heterogeneous spontaneous mammary tumor animal model of Kunming mice was generated. The purpose of this study was to characterize the spontaneous mammary tumor model. Histopathologically, invasive nodular masses of pleomorphic tubular neoplastic epithelial cells invaded fibro-vascular stroma, adjacent dermis and muscle tissue. Metastatic spread through blood vessel into liver and lungs was observed by hematoxylin eosin staining. No estrogen receptor (ER) or progesterone receptor (PR) immunoreactivity was detected in their associated malignant tumors, human epidermal growth factor receptor-2 (HER-2) protein weak expression was found by immunohistochemistry. High expression of vascular endothelial growth factor (VEGF), moderate or high expression of c-Myc and cyclin D1 were observed in tumor sections at different stages (2, 4, 6 and 8 weeks after cancer being found) when compared with that of the normal mammary glands. The result showed that the model is of an invasive ductal carcinoma. Remarkably in the mouse model, ER and PR-negative and HER2 weak positivity are observed. The high or moderate expressions of breast cancer markers (VEGF, c-Myc and cyclin D1) in mammary cancer tissue change at different stages. To our knowledge, this is the first report of a spontaneous mammary model displaying colony-strain, outbred mice. This model will be an attractive tool to understand the biology of anti-hormonal breast cancer in women. PMID:25230850

  16. Regeneration of Bovine Mammary Gland in Immunodeficient Mice by Transplantation of Bovine Mammary Epithelial Cells Mixed with Matrigel

    PubMed Central

    Park, Hyun Jung; Lee, Won Young; Jeong, Ha Yeon; Song, Hyuk

    2016-01-01

    Background and Objectives With the global demand for dairy protein for consumption growing annually, there has been increasing activity in the research field of dairy protein synthesis and production. From a manipulation perspective, it is more difficult to use live cattle for laboratory studies on the production of milk as well as of dairy protein such as casein, as compared with using laboratory animals like rodents. Therefore, we aimed to develop a mouse model of bovine mammary alveolar ducts for laboratory-scale studies. We studied the formation of the bovine mammary gland ductal structure by transplanting the MAC-T bovine alveolar cell line into mice. Methods and Results MAC-T cells (1×107) were suspended in Matrigel and injected into the dorsal tissue of 8-week-old male BALB/C nude mice. Histological analysis of tissue dissected from the MAC-T cell-transplanted mice after 6 weeks showed the typical morphology of the tubuloalveolar female gland, as well as glands made up of branching ducts that were surrounded by smooth muscle with small alveoli budding off the ducts. In addition, the epithelial markers CK14 and CK18 were expressed within the duct-like structure. Prolactin was detected in the duct interior in these CK14+ and CK18+ cells but not in the non-transplanted MAC-T cells. Conclusions These results showed that duct-like tissue had been successfully formed after 6 weeks of transplantation of the CK14+ and CK18+ MAC-T cells into mice dorsal tissue. This mouse model will be a useful tool for further research on the bovine mammary gland. PMID:27788570

  17. Ink4a/Arf(-/-) and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f(-) quiescent cells.

    PubMed

    Kai, K; Iwamoto, T; Kobayashi, T; Arima, Y; Takamoto, Y; Ohnishi, N; Bartholomeusz, C; Horii, R; Akiyama, F; Hortobagyi, G N; Pusztai, L; Saya, H; Ueno, N T

    2014-01-23

    Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (-/-) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf(-/-) mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (TNBC) (for example, pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f(-) quiescent cells and CD49f(+)cells. Contrary to our expectation, CD49f(-) quiescent cells had high tumor-initiating potential and CD49f(+)cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f(-) quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human TNBC.

  18. Novel radiation response genes identified in gene-trapped MCF10A mammary epithelial cells.

    PubMed

    Malone, Jennifer; Ullrich, Robert

    2007-02-01

    We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2-30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

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

    PubMed

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

    2016-06-30

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

  20. From milk to malignancy: the role of mammary stem cells in development, pregnancy and breast cancer.

    PubMed

    Tiede, Benjamin; Kang, Yibin

    2011-02-01

    Adult stem cells of the mammary gland (MaSCs) are a highly dynamic population of cells that are responsible for the generation of the gland during puberty and its expansion during pregnancy. In recent years significant advances have been made in understanding how these cells are regulated during these developmentally important processes both in humans and in mice. Understanding how MaSCs are regulated is becoming a particularly important area of research, given that they may be particularly susceptible targets for transformation in breast cancer. Here, we summarize the identification of MaSCs, how they are regulated and the evidence for their serving as the origins of breast cancer. In particular, we focus on how changes in MaSC populations may explain both the increased risk of developing aggressive ER/PR(-) breast cancer shortly after pregnancy and the long-term decreased risk of developing ER/PR(+) tumors.

  1. DNA measurement and immunohistochemical characterization of epithelial and mesenchymal cells in canine mixed mammary tumours: putative evidence for a common histogenesis.

    PubMed

    Gärtner, F; Geraldes, M; Cassali, G; Rema, A; Schmitt, F

    1999-07-01

    DNA measurement by image cytometry, and a detailed immunohistochemical study using monoclonal antibodies directed against different human cytokeratin types, muscle-specific actin, vimentin and S100 protein were carried out on normal canine mammary tissue (n =4), benign canine mammary mixed tumours (n =20) and malignant canine mammary mixed tumours (n =13). The results showed that ductal and alveolar luminal cells in normal and neoplastic tissue were immunoreactive with CAM5.2 and AE1/AE3 antibodies recognizing human keratins.Basal/myoepithelial cells were clearly differentiated from ductal and alveolar epithelial cells, since the latter only expressed cytokeratins, whereas the former also expressed vimentin and muscle-specific actin. This immunohistochemical study showed that there is loss of expression of muscle-specific actin and cytokeratins in areas of myoepithelial proliferation, and enhanced expression of vimentin and S100 protein in proliferative areas with osseous and/or chondroid metaplasia. The ploidy studies revealed that 20% (4/20) of benign and 54% (7/13) of malignant mixed tumours of canine mammary gland were aneuploid and that the epithelial and myoepithelial components of the mixed tumours had identical DNA content. Our results reinforce the role of myoepithelial cells in mesenchymal metaplasia in mixed mammary tumours and suggest the possibility of a common origin of both components from a totipotential stem cell with capacity for divergent differentiation.

  2. The Transcriptional Repressor ZNF503/Zeppo2 Promotes Mammary Epithelial Cell Proliferation and Enhances Cell Invasion*

    PubMed Central

    Shahi, Payam; Slorach, Euan M.; Wang, Chih-Yang; Chou, Jonathan; Lu, Angela; Ruderisch, Aline; Werb, Zena

    2015-01-01

    The NET (nocA, Nlz, elB, TLP-1) subfamily of zinc finger proteins is an important mediator during developmental processes. The evolutionary conserved zinc finger protein ZNF503/Zeppo2 (zinc finger elbow-related proline domain protein 2, Zpo2) plays critical roles during embryogenesis. We found that Zpo2 is expressed in adult tissue and examined its function. We found that ZPO2 is a nuclearly targeted transcriptional repressor that is expressed in mammary epithelial cells. Elevated Zpo2 levels increase mammary epithelial cell proliferation. Zpo2 promotes cellular invasion through down-regulation of E-cadherin and regulates the invasive phenotype in a RAC1-dependent manner. We detect elevated Zpo2 expression during breast cancer progression in a MMTV-PyMT transgenic mouse model. Tumor transplant experiments indicated that overexpression of Zpo2 in MMTV-PyMT mammary tumor cell lines enhances lung metastasis. Our findings suggest that Zpo2 plays a significant role in mammary gland homeostasis and that deregulation of Zpo2 may promote breast cancer development. PMID:25538248

  3. Isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture.

    PubMed

    Xu, Kun; Buchsbaum, Rachel J

    2012-04-30

    While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors(1-2), much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension(3-4). Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture(3). While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis. The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized(4). Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells(5). Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals(6). Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors(7). Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion(8-15). We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three

  4. Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

    PubMed

    Cravero, Diego; Diego, Cravero; Martignani, Eugenio; Eugenio, Martignani; Miretti, Silvia; Silvia, Miretti; Macchi, Elisabetta; Elisabetta, Macchi; Accornero, Paolo; Paolo, Accornero; Baratta, Mario; Mario, Baratta

    2014-10-01

    The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

  5. PREPARATION OF EPITHELIAL AND MESENCHYMAL STEM CELLS FROM MURINE MAMMARY GLAND

    PubMed Central

    Guest, I.; Ilic, Z.; Ma, J.

    2011-01-01

    The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for this remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44 and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumour development. PMID:22058055

  6. Effects of iron oxide nanoparticles on biological responses and MR imaging properties in human mammary healthy and breast cancer epithelial cells.

    PubMed

    Zhang, Qin; Rajan, Sunder S; Tyner, Katherine M; Casey, Brendan J; Dugard, Christopher K; Jones, Yvonne; Paredes, Angel M; Clingman, Chekesha S; Howard, Paul C; Goering, Peter L

    2016-07-01

    Superparamagnetic iron oxide nanoparticles (SPIONs, diameters >50 nm) have received great attention due to their promising use as magnetic resonance imaging (MRI) contrast agents. In this study, we evaluated the cellular uptake and biological responses in vitro of ultrasmall SPIONs (USPIONs, diameters < 50 nm). We compared the cellular responses between breast epithelia isolated from healthy and breast cancer donors after exposure to carboxy-terminated USPIONs (10 and 30 nm PEG-coated, 10 and 30 nm non-PEG-coated). The particles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS) and gel electrophoresis. Cellular interactions with USPIONs were assessed by confocal microscopy and TEM. Cellular uptake of USPIONs was quantified using ICP-MS. Cell viability was measured by MTT and neutral red uptake assays. T2* weighted MRI scans were performed using a 7T scanner. Results demonstrated that cell association/internalization of USPIONs was size- and surface coating-dependent (PEG vs. non-PEG), and higher cellular uptake of 10 and 30 nm non-coated particles was observed in both cell types compared with PEG-coated particles. Cell uptake for 10 and 30 nm non-coated particles was higher in cancer cells from two of three tested donors compared to healthy cells from three donors. There was no significant cytotoxicity observed for all tested particles. Significantly enhanced MRI contrast was observed following exposure to 10 and 30 nm non-coated particles compared to PEG-coated particles in both cell types. In comparison, cancer cells showed more enhanced MRI signals when compared to normal cells. The data indicate that cell responses following exposure to USPIONs are dependent on particle properties. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1032-1042, 2016.

  7. Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

    PubMed

    Keshava, Channa; Divi, Rao L; Einem, Tracey L; Richardson, Diana L; Leonard, Sarah L; Keshava, Nagalakshmi; Poirier, Miriam C; Weston, Ainsley

    2009-03-01

    We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.

  8. Evaluation of Leucocyte Functions Six Years after Tumour Autograft in Human Mammary Cancer

    PubMed Central

    Anderson, J. Maxwell; Kelly, F.; Wood, Suzanne E.; Rodger, K. D.; Freshney, R. Ian

    1973-01-01

    Mammary cancer directed and nonspecific immunoassays were made in 3 groups of female patients. One group had primary mammary cancer treated by mastectomy and postoperative radiotherapy plus an autograft of irradiated tumour (AIT) 40-66 months previously. A second age-matched group had mammary cancer comparable to the first group in clinical presentation and treatment except that no AIT was given. The third group consisted of non-cancer-bearing age-matched females. The migration of leucocytes from autografted patients was significantly inhibited in the presence of allogeneic mammary cancer cells from a standardized panel, compared with leucocytes from either non-autograft patients or non-cancer bearers. Selected data from a lymphocyte cytotoxicity test revealed a significantly greater kill of allogeneic mammary cancer target cells by autograft lymphocytes than by those of other groups. These indications of increased cancer directed cell mediated immunity in respect of sensitivity and toxicity in association with AIT require further elucidation under strictly controlled conditions. PMID:4807858

  9. Mammary stem cells and parity-induced breast cancer protection- new insights.

    PubMed

    Dall, Genevieve; Risbridger, Gail; Britt, Kara

    2016-02-22

    Parity (childbearing) significantly decreases a woman's risk of breast cancer and the protective effect is greater if the woman is younger and has more children. The mechanism/s of parity-induced protection are not known. Although several factors are postulated to play a role, we discuss how a reduction in the number of mammary stem cells (MaSCs) may lead to a reduction in breast cancer risk in parous women. Firstly we review the epidemiology linking childbearing to reduced breast cancer risk and discuss how additional births, a young age at first full term birth, and breastfeeding impact the protection. We then detail the mouse and human studies implicating MaSC in parity induced protection and the in-vivo work being performed in mice to directly investigate the effect of parity on MaSC. Finally we discuss the transplant and lineage tracing experiments assessing MaSC activity according to parity and the need to define if MaSC are indeed more carcinogen sensitive than mature mammary epithelial cells. Continuing and future studies attempting to define the parity induced mechanisms will aid in the development of preventative therapies.

  10. Wwox inactivation enhances mammary tumorigenesis.

    PubMed

    Abdeen, S K; Salah, Z; Maly, B; Smith, Y; Tufail, R; Abu-Odeh, M; Zanesi, N; Croce, C M; Nawaz, Z; Aqeilan, R I

    2011-09-08

    Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3-4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (Wwox(C3H)+/-) and incidence of mammary tumor formation was evaluated. Although 50% of the female Wwox(C3H)+/- mice developed mammary carcinomas, only 7% of Wwox(C3H)+/+ mice did. Intriguingly, mammary tumors in Wwox(C3H)+/- mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-α (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorage-independent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in Wwox(C3H)+/- mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.

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

    SciTech Connect

    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 hitherto 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.

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

    DTIC Science & Technology

    2011-02-01

    cells was established by transplantation experiments in mice. For example, a functioning ductal tree can be regenerated using very few transplanted...counterstained with DAPI (blue) and imaged for the expression of both Tomato (red) and GFP (green) proteins. 9 We have collected and transplanted Lin...determine the expression of cre recombinase in the mammary epithelium. We can detect EGFP but not Tomato in the mammary epithelium of these animals

  13. Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro

    PubMed Central

    McGrath, Charles M.; Nandi, S.; Young, Lawrence

    1972-01-01

    Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called “domes.” MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly

  14. Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

    PubMed Central

    Li, Guangyuan; Hayward, Isaac N.; Jenkins, Brittany R.; Rothfuss, Heather M.; Young, Coleman H.; Nevalainen, Marja T.; Muth, Aaron; Thompson, Paul R.; Navratil, Amy M.; Cherrington, Brian D.

    2016-01-01

    Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation. PMID:26799659

  15. Characterization of mammary adenocarcinomas in male rats after N-methyl-N-nitrosourea exposure--Potential for human male breast cancer model.

    PubMed

    Yoshizawa, Katsuhiko; Yuki, Michiko; Kinoshita, Yuichi; Emoto, Yuko; Yuri, Takashi; Shikata, Nobuaki; Elmore, Susan A; Tsubura, Airo

    2016-05-01

    The frequency of breast cancer in men is extremely rare, reported to be less than 1% and there is currently no available animal model for male mammary tumors. We compared the characteristics of various immunohistochemical markers in N-methyl-N-nitrosourea (MNU)-induced mammary adenocarcinomas in male and female Crj:CD(SD)IGS rats including: estrogen receptor α (ER), progesterone receptor (PgR), androgen receptor (AR), receptor tyrosine-protein kinase erbB-2 (HER2), GATA binding protein 3 (GATA3), and proliferating cell nuclear antigen (PCNA). Female mammary adenocarcinomas were strongly positive in the nuclei of tumor cells for PCNA and ER (100%) with only 60% and 53% expressing PgR and GATA3, respectively. 100% of male adenocarcinomas also exhibited strongly positive expression in the nuclei of tumor cells for PCNA, with 25% expressing AR and only 8% showing positivity for ER. Male carcinomas did not express PgR or GATA3 and none of the tumors, male or female, were positive for HER2. Based on the observed ER and PgR positivity and HER2 negativity within these tumors, MNU-induced mammary adenocarcinomas in female rats appear to be hormonally dependent, similar to human luminal A type breast cancer. In contrast, MNU-induced mammary adenocarcinomas in male rats showed no reactivity for ER, PgR, HER2 or GATA3, suggesting no hormonal dependency. Both male and female adenocarcinomas showed high proliferating activity by PCNA immunohistochemistry. Based on our literature review, human male breast cancers are mainly dependent on ER and/or PgR, therefore the biological pathogenesis of MNU-induced male mammary cancer in rats may differ from that of male breast cancer in humans.

  16. Erythropoietin Induces an Epithelial to Mesenchymal Transition-like Process in Mammary Epithelial Cells MCF10A.

    PubMed

    Ordoñez-Moreno, Alejandra; Rodriguez-Monterrosas, Cecilia; Cortes-Reynosa, Pedro; Perez-Carreon, Julio Isael; Perez Salazar, Eduardo

    2017-03-01

    Anemia is associated with chemotherapy treatment in cancer patients. Erythropoietin (EPO) has been used to treat anemia of cancer patients, because it stimulates erythropoiesis. However, treatment of breast cancer patients with EPO has been associated with poor prognosis and decrease of survival. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to a mesenchymal state. It has been implicated in tumor progression, because epithelial cells acquire the capacity to execute the multiple steps of invasion/metastasis process. However, the role of EPO on EMT process in human mammary epithelial cells has not been studied. In the present study, we demonstrate that EPO promotes a decrease of E-cadherin expression, an increase of N-cadherin, vimentin and Snail2 expression, activation of FAK and Src kinases and an increase of MMP-2 and MMP-9 secretions. Moreover, EPO induces an increase of NFκB DNA binding activity, an increase of binding of p50 and p65 NFκB subunits to Snail1 promoter, migration and invasion in mammary non-tumorigenic epithelial cells MCF10A. In summary, these findings demonstrate, for the first time, that EPO induces an EMT-like process in mammary non-tumorigenic epithelial cells. This article is protected by copyright. All rights reserved.

  17. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    PubMed Central

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

    2016-01-01

    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  18. Role of P13 Kinase Signaling Pathways in Polarity Determination of Human Mammary Epithelial Cells Grown in Three-Dimensional Extracellular Matrix

    DTIC Science & Technology

    2004-09-01

    because cells died once they overexpressed PTEN-GFP construct. The primary consequence of P13K activation is the generation of PIP3 in the membrane...grow in anchorage- independent assays (Figure 3). Our results are consistent with another recent study in that active Akt, when overexpressed ...grown in methyl downstream of P13K might exist to cellulose for 3 weeks and micrographs were control polarity, taken. Now came the question as to which

  19. The effects of cell death-inducing DNA fragmentation factor-α-like effector C on milk lipid synthesis in mammary glands of dairy cows.

    PubMed

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

    2017-03-08

    Adequate lipid synthesis by the mammary gland during lactation is essential for the survival of mammalian offspring. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) is a lipid droplet-associated protein and functions to promote lipid accumulation and inhibit lipolysis in mice and human adipocytes. However, the function of CIDEC in regulation of milk lipid synthesis in dairy cow mammary gland remains largely unknown. In this study, 6 multiparous Holstein cows (parity = 3) in early lactation were allocated to high-fat milk (milk yield 33.9 ± 2.1 kg/d, milk fat >3.5%, n = 3) and low-fat milk (milk yield 33.7 ± 0.5 kg/d, milk fat <3.5%, n = 3) groups according to their milk fat content. Lactating cows were slaughtered at 90 d in milk and mammary tissues were collected to detect CIDEC localization. Immunofluorescence staining of sections of lactating mammary glands with high- and low-fat milk showed that CIDEC was expressed in the cytoplasm of epithelial cells and localized to lipid droplets. Lipid droplets and CIDEC protein were also detected in isolated lactating mammary epithelial cells of dairy cows. Immunostaining of CIDEC in isolated mammary epithelial cells also confirmed its presence in the nucleus. The knockdown of CIDEC in cultured bovine mammary epithelial cells decreased milk lipid content and reduced expression of genes associated with mammary de novo fatty acid synthesis, short- and long-chain intracellular fatty acid activation, triacylglycerol synthesis, and transcription regulation. These genes included those for acetyl-CoA carboxylase (ACC, -60%), fatty acid synthase (FASN, -65%), acyl-CoA synthetase short-chain family member 2 (ACSS2, -50%), acyl-CoA synthetase long-chain family member 1 (ACSL1, -30%), diacylglycerol acyltransferase 1 (DGAT1, -60%), sterol regulatory element-binding protein 1 (SREBP1, -45%), and SREBP cleavage activating protein (SCAP, -66%). Conversely, in cells overexpressing CIDEC, triacylglycerol content

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

    PubMed Central

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

    2014-01-01

    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. What's new? Can eating fermented foods like yogurt ward off cancer? Recent studies have suggested it's possible. To find out how, these authors isolated the probiotic bacteria involved in fermentation and fed them to mice that were

  1. On the possible role of mammary-derived growth hormone in human breast cancer.

    PubMed

    Thijssen, Jos H H

    2009-12-01

    The incidence of breast cancer has risen worldwide, especially in countries where it used to be low, very probably as a result of economic prosperity and changes in life-style. In women, the available data have resulted in the concept of progression from normal breast development to cancer through precursor lesions sensitive to hormones and growth factors that can be produced locally in the mammary gland, acting as paracrine or autocrine stimulating agents. The local endocrine environment in the breast can be different from the situation in the circulation. In the dog, growth hormone (GH) can be produced locally in the mammary glands and its production can be stimulated by progestins. This GH probably plays a paracrine role in the progesterone-induced proliferation and differentiation of mammary epithelium. There is increasing evidence that the local mammary progestin/GH-axis is operational not only in dogs but also in human breast cancer. No data are yet available on the production of mammary-derived GH in women.

  2. The co-factor of LIM domains (CLIM/LDB/NLI) maintains basal mammary epithelial stem cells and promotes breast tumorigenesis.

    PubMed

    Salmans, Michael L; Yu, Zhengquan; Watanabe, Kazuhide; Cam, Eric; Sun, Peng; Smyth, Padhraic; Dai, Xing; Andersen, Bogi

    2014-07-01

    Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

  3. Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation.

    PubMed

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

    2007-09-01

    We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females. PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ(+)) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ(+) cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. LacZ(+) cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ(+) mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse epithelial cell fates.

  4. The effect of hyposmotic and isosmotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinar cells.

    PubMed

    Shennan, D B; Grant, A C G; Gow, I F

    2002-04-01

    The effect of hyposmotic and isosmotic cell swelling on the free intracellular calcium concentration ([Ca2+]i) in rat mammary acinar cells has been examined using the fura-2 dye technique. Ahyposmotic shock (40% reduction) increased the [Ca2+]i in rat mammary acinar cells in a fashion which was transient; the [Ca2+]i returned to a value similar to that found under isomotic conditions within 180 sec. The increase in the [Ca2+]i was dependent upon the extent of the osmotic shock. The hyposmotically-activated increase in the [Ca2+]i could not be attributed to a reduction in extracellular Na+ or a change in the ionic strength of the incubation medium. Thapsigargin (1 microM) enhanced the hyposmotically-activated increase in the [Ca2+]i. Isosmotic swelling of rat mammary acinar cells, using urea, had no significant effect on the [Ca2+]i. Similarly, a hyperosmotic shock did not affect the [Ca2+]i in rat mammary acinar cells. It appears that the effect of cell swelling on the [Ca2+]i in rat mammary acinar cells depends on how the cells are swollen (hyposmotic vs. isosmotic). This finding may have important physiological implications given that it is predicted that mammary cell volume will change in vivo under isomotic conditions.

  5. Hops (Humulus lupulus) inhibits oxidative estrogen metabolism and estrogen-induced malignant transformation in human mammary epithelial cells (MCF-10A).

    PubMed

    Hemachandra, L P; Madhubhani, P; Chandrasena, R; Esala, P; Chen, Shao-Nong; Main, Matthew; Lankin, David C; Scism, Robert A; Dietz, Birgit M; Pauli, Guido F; Thatcher, Gregory R J; Bolton, Judy L

    2012-01-01

    Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the-counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor-mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be significantly inhibited by hops (5 μg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN.

  6. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

    PubMed Central

    Dzik, Luciana M.; Iglesia, Rebeca P.; Cruz, Mário C.; Zelanis, André; de Siqueira, Adriane S.; Serrano, Solange M.T.; Goldberg, Gary S.; Jaeger, Ruy G.; Freitas, Vanessa M.

    2016-01-01

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment. PMID:27374178

  7. Control of mammary myoepithelial cell contractile function by α3β1 integrin signalling.

    PubMed

    Raymond, Karine; Cagnet, Stéphanie; Kreft, Maaike; Janssen, Hans; Sonnenberg, Arnoud; Glukhova, Marina A

    2011-05-18

    In the functionally differentiated mammary gland, basal myoepithelial cells contract to eject the milk produced by luminal epithelial cells from the body. We report that conditional deletion of a laminin receptor, α3β1 integrin, from myoepithelial cells leads to low rates of milk ejection due to a contractility defect but does not interfere with the integrity or functional differentiation of the mammary epithelium. In lactating mammary gland, in the absence of α3β1, focal adhesion kinase phosphorylation is impaired, the Rho/Rac balance is altered and myosin light-chain (MLC) phosphorylation is sustained. Cultured mammary myoepithelial cells depleted of α3β1 contract in response to oxytocin, but are unable to maintain the state of post-contractile relaxation. The expression of constitutively active Rac or its effector p21-activated kinase (PAK), or treatment with MLC kinase (MLCK) inhibitor, rescues the relaxation capacity of mutant cells, strongly suggesting that α3β1-mediated stimulation of the Rac/PAK pathway is required for the inhibition of MLCK activity, permitting completion of the myoepithelial cell contraction/relaxation cycle and successful lactation. This is the first study highlighting the impact of α3β1 integrin signalling on mammary gland function.

  8. Differentiation of the mammary epithelial cell during involution: implications for breast cancer.

    PubMed

    Monks, Jenifer; Henson, Peter M

    2009-06-01

    That milk secretion is not the final differentiated state of the mammary alveolar cells is a relatively new concept. Recent work has suggested that secreting, mammary epithelial cells (MECs) have another function to perform before they undergo cell death in the involuting mammary gland. That is, they help in the final clearance and breakdown of their neighboring cells (and likely residual milk as well.) They become, for a short time, amateur phagocytes, or efferocytes, and then are believed to die and be cleared themselves. Although relatively little study has been made of this change in the functional state of the MEC, nevertheless we may speculate from the involution literature, and extend findings from other systems of apoptotic cell clearance, on some of the mechanisms involved. And with the finding that involution may represent a unique susceptibility window for the progression of metastatic breast cancer, we may suggest areas for future research along these lines as well.

  9. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    SciTech Connect

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  10. Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells[S

    PubMed Central

    Hsieh, Chang-Wen; Huang, Charles; Bederman, Ilya; Yang, Jianqi; Beidelschies, Michelle; Hatzoglou, Maria; Puchowicz, Michelle; Croniger, Colleen M.

    2011-01-01

    Previously, we have shown that Pck1 expression in mammary gland adipocytes and white adipose tissue maintains triglyceride stores through glyceroneogenesis, and these lipids were used for synthesis of milk triglycerides during lactation. Reduced milk triglycerides during lactation resulted in patterning of the newborn for insulin resistance. In this study, the role of Pck1 in mammary gland epithelial cells was analyzed. The developmental expression of Pck1 decreased in isolated mouse mammary gland epithelial cells through development and during lactation. Using HC11, a clonal mammary epithelial cell line, we found that both Janus kinase 2 signal transducers and activators of transcription 5 and the AKT pathways contributed to the repression of Pck1 mRNA by prolactin. These pathways necessitate three accessory factor regions of the Pck1 promoter for repression by prolactin. Using [U-13C6]glucose, [U-13C3]pyruvate, and [U-13C3]glycerol in HC11 cells, we determined that Pck1 functions in the pathway for the conversion of gluconeogenic precursors to glucose and contributes to glycerol-3-phosphate synthesis through glyceroneogenesis. Therefore, Pck1 plays an important role in both the mammary gland adipocytes and epithelial cells during lactation. PMID:21504969

  11. α2-Adrenoceptor action on cell proliferation and mammary tumour growth in mice

    PubMed Central

    Bruzzone, A; Piñero, C Pérez; Castillo, L F; Sarappa, M G; Rojas, P; Lanari, C; Lüthy, I A

    2008-01-01

    Background and purpose: Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described α2-adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on α2-adrenoceptors in a reliable model of breast cancer. Experimental approach: The expression of α2-adrenoceptors was confirmed by immunocytochemistry, immunofluorescence and reverse transcription-PCR in the mouse mammary tumour cell line MC4-L5. Proliferation was assessed by [3H]thymidine incorporation and tumours were measured daily. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling. Key results: Incubation for 2 days with α2-adrenoceptor agonists (clonidine and dexmedetomidine) significantly enhanced proliferation of the mouse mammary tumour cell line MC4-L5. These agonists also significantly stimulated tumour growth of the progestin-dependent tumour C4-HD even in the presence of medroxyprogesterone acetate (MPA). In every tumour tested (C4-HD, CC4-2-HD and CC4-3-HI), regardless of MPA sensitivity, clonidine significantly enhanced tumour growth in the absence of MPA. The α2-adrenoceptor antagonists, yohimbine and rauwolscine, completely reversed the effects of clonidine. However, the group receiving yohimbine alone showed a nonsignificant but constant increase in tumour growth, whereas rauwolscine alone diminished tumour growth significantly, behaving as a reverse agonist. In CC4-3-HI tumours, rauwolscine treatment enhanced apoptosis and diminished the mitotic index, whereas clonidine had the inverse effect. Conclusions and implications: α2-Adrenoceptor agonists enhanced tumour growth and rauwolscine behaved in vivo as a reverse agonist, suggesting that it may be tested for adjuvant treatment. PMID:18604234

  12. CD44+/CD24- Cancer Stem Cells Are Associated With Higher Grade of Canine Mammary Carcinomas.

    PubMed

    Im, K S; Jang, Y G; Shin, J I; Kim, N H; Lim, H Y; Lee, S M; Kim, J H; Sur, J H

    2015-11-01

    The CD44+/CD24- phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24- phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)-negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24- phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.

  13. Genetic Manipulation Of Mammary Stem Cells to Reconcile Tumor Stem Cell Theory with Breast Cancer Heterogeneity

    DTIC Science & Technology

    2010-07-01

    analyzed by reverse transcription- polymerase chain reaction analysis (RT-PCR). GFP mRNA was not found in ΔN-p63-eGFPcre mammary gland. In the K14...transgene were selected by exploiting a neomycin resistance gene. The cells were then infected with an adenovirus bearing a gene for Cre- recombinase ...Successful genomic recombination was detected by polymerase chain reaction (PCR) analysis of genomic DNA. RT-PCR analysis was used to determine

  14. Expression, localization, and functional model of cholesterol transporters in lactating and nonlactating mammary tissues of murine, bovine, and human origin.

    PubMed

    Mani, Orlando; Körner, Meike; Sorensen, Martin T; Sejrsen, Kristen; Wotzkow, Carlos; Ontsouka, Corneille E; Friis, Robert R; Bruckmaier, Rupert M; Albrecht, Christiane

    2010-08-01

    Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species.

  15. Anti-influenza neuraminidase inhibitor oseltamivir phosphate induces canine mammary cancer cell aggressiveness.

    PubMed

    de Oliveira, Joana T; Santos, Ana L; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J; Vasconcelos, M Helena; Oliveira, Maria José; Reis, Celso A; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness.

  16. Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

    PubMed Central

    de Oliveira, Joana T.; Santos, Ana L.; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J.; Vasconcelos, M. Helena; Oliveira, Maria José; Reis, Celso A.; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness. PMID:25850034

  17. Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

    PubMed Central

    Bersani, Francesca; Quaglino, Elena; Martignani, Eugenio; Baratta, Mario

    2012-01-01

    Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs. PMID:23028720

  18. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

    SciTech Connect

    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  19. Fibroblast growth factor receptor signaling is essential for normal mammary gland development and stem cell function.

    PubMed

    Pond, Adam C; Bin, Xue; Batts, Torey; Roarty, Kevin; Hilsenbeck, Susan; Rosen, Jeffrey M

    2013-01-01

    Fibroblast growth factor (FGF) signaling plays an important role in embryonic stem cells and adult tissue homeostasis, but the function of FGFs in mammary gland stem cells is less well defined. Both FGFR1 and FGFR2 are expressed in basal and luminal mammary epithelial cells (MECs), suggesting that together they might play a role in mammary gland development and stem cell dynamics. Previous studies have demonstrated that the deletion of FGFR2 resulted only in transient developmental defects in branching morphogenesis. Using a conditional deletion strategy, we investigated the consequences of FGFR1 deletion alone and then the simultaneous deletion of both FGFR1 and FGFR2 in the mammary epithelium. FGFR1 deletion using a keratin 14 promoter-driven Cre-recombinase resulted in an early, yet transient delay in development. However, no reduction in functional outgrowth potential was observed following limiting dilution transplantation analysis. In contrast, a significant reduction in outgrowth potential was observed upon the deletion of both FGFR1 and FGFR2 in MECs using adenovirus-Cre. Additionally, using a fluorescent reporter mouse model to monitor Cre-mediated recombination, we observed a competitive disadvantage following transplantation of both FGFR1/R2-null MECs, most prominently in the basal epithelial cells. This correlated with the complete loss of the mammary stem cell repopulating population in the FGFR1/R2-attenuated epithelium. FGFR1/R2-null MECs were partially rescued in chimeric outgrowths containing wild-type MECs, suggesting the potential importance of paracrine mechanisms involved in the maintenance of the basal epithelial stem cells. These studies document the requirement for functional FGFR signaling in mammary stem cells during development.

  20. Contribution of xanthine oxidoreductase to mammary epithelial and breast cancer cell differentiation in part modulates inhibitor of differentiation-1.

    PubMed

    Fini, Mehdi A; Monks, Jenifer; Farabaugh, Susan M; Wright, Richard M

    2011-09-01

    Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

  1. Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines.

    PubMed

    Michishita, Masaki; Akiyoshi, Rui; Yoshimura, Hisashi; Katsumoto, Takuo; Ichikawa, Hitoshi; Ohkusu-Tsukada, Kozo; Nakagawa, Takayuki; Sasaki, Nobuo; Takahashi, Kimimasa

    2011-10-01

    There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

  2. Feasibility and Technical Considerations of Mammary Ductoscopy in Human Mastectomy Specimens.

    PubMed

    Dietz, Jill R.; Kim, Julian A.; Malycky, Jan L.; Levy, Lawrence; Crowe, Joseph

    2000-05-01

    Recent advances in endoscopic technology have made visualization of human mammary ducts possible. The purpose of this study was to assess the feasibility and technical factors influencing the ability to successfully visualize the epithelium of the human mammary ductal system. Lacrimal duct probes were used to dilate nipple orifices to 1.2 mm on 42 mastectomy specimens. The Depth of Field Imaging Micro-Minimally Invasive (DOFI(R) MMI) system consisting of a 1.2 mm rigid ductoscope with a 350 &mgr;m working channel was introduced into mammary ducts under air insufflation or saline irrigation. At least one major duct could be dilated and cannulated in all 42 specimens. Visualization of the proximal duct was accomplished in 34 of 42 (81%) specimens, whereas more extensive navigation through the distal subsegmental ducts was achieved in 22 of 42 (52%) specimens. Ductoscopy into the terminal ducts was accomplished in all patients with a previous history of nipple discharge or discharge at the time of the procedure (10 of 10). In three patients with no history of nipple discharge prior to ductoscopy, incidental papillomas were discovered and confirmed by the pathologist. In conclusion, mammary ductoscopy is technically feasible and may have an application as an additional diagnostic modality for patients with pathologic nipple discharge.

  3. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  4. miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells.

    PubMed

    Liao, Yalin; Du, Xiaogu; Lönnerdal, Bo

    2010-09-01

    Lactoferrin (Lf) is an abundantly expressed protein in human milk. Lactoferrin exhibits several important biological functions, and its expression is regulated by multiple environmental factors. Cellular endogenous factors, however, have not been extensively studied with regard to lactoferrin gene expression. In this study, we showed that lactoferrin gene expression and function are directly targeted by miR-214 in HC11 and MCF7 cells. In the lactoferrin mRNA 3 prime untranslated region (UTR) of human, mouse, rat, pig, bovine, camel, and goat species, there is a conserved region that perfectly matches the seed region of miR-214. Transfection of miR-214 mimic in HEK293 cells dose-dependently inhibited the activity of pGL3-control vector containing lactoferrin mRNA 3 prime UTR downstream of the luciferase gene. In HC11 cells, miR-214 overexpression inhibited the induction of lactoferrin expression by beta -estradiol (E2) and dexamethasone-prolactin-insulin (DPI). Furthermore, in MCF7 cells, overexpression of miR-214 markedly decreased lactoferrin expression (P lt 0.05), and inhibition of endogenous miR-214 expression increased lactoferrin expression and cellular apoptotic activities (P lt 0.05). In summary, our data showed that miR-214 is directly involved in lactoferrin expression and lactoferrin mediated cancer susceptibility (proapoptotic activities) in mammary epithelial cells.

  5. The role of unfolded protein response in differentiation of mammary epithelial cells.

    PubMed

    Tsuchiya, Megumi; Koizumi, Yumiko; Hayashi, Satoko; Hanaoka, Miyuki; Tokutake, Yukako; Yonekura, Shinichi

    2017-03-18

    The accumulation of misfolded proteins in the ER provokes ER stress by increasing the demand for energy, chaperones, and other proteins that are needed to fold client proteins or to degrade unfoldable secretory cargo. This stress activates a signaling network called the unfolded protein response (UPR). However, recent accumulated data suggested that the UPR also provides important signals for regulating cell differentiation and maturation. However, the relationship between UPR and mammary gland development has not been fully elucidated. To define the involvement of the UPR in mammary gland development, mammary glands were collected from non-pregnant mice, at days 5, 10 and 15 of pregnancy, at days 1 and 7 of lactation, and the expression patterns of UPR-related genes were determined by real-time PCR. We found that the mRNA expression of ATF4 and XBP1 significant increased during pregnancy. Moreover, we found that both ATF4 and XBP1 proteins are expressed in mammary epithelial cells by immunohistological analysis. In order to know the role of ATF4 and XBP1 in the differentiation of mammary epithelial cell, we performed gene knockdown experiment using HC11 cells. We found that ATF4 or XBP1 knockdown suppressed the mRNA expression of beta-casein and lactogenic hormone receptor in differentiating HC11 cells. Our results demonstrate that XBP1 and ATF4, which are UPR-related transcription factors, directly or indirectly participate in cell differentiation mechanisms through the regulation of the expression of lactogenic hormone receptors in mouse mammary epithelial cells.

  6. Stem/progenitor cells in non-lactating versus lactating equine mammary gland.

    PubMed

    Spaas, Jan H; Chiers, Koen; Bussche, Leen; Burvenich, Christian; Van de Walle, Gerlinde R

    2012-11-01

    The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation, and involution. Based on the facts that (i) mammary stem/progenitor cells (MaSC) are proposed to be the driving forces behind mammary growth and function and (ii) variation exists between mammalian species with regard to physiological and pathological functioning of this organ, we believe that studying MaSC from different mammals is of great comparative interest. Over the years, important data has been gathered on MaSC of men and mice, although knowledge on MaSC in other mammals remains limited. Therefore, the aim of this work was to isolate and characterize MaSC from the mammary gland of horses. Hereby, our salient findings were that the isolated equine cells met the 2 in vitro hallmark properties of stem cells, namely the ability to self-renew and to differentiate into multiple cell lineages. Moreover, the cells were immunophenotyped using markers for CD29, CD44, CD49f, and Ki67. Finally, we propose the mammosphere assay as a valuable in vitro assay to study MaSC during different physiological phases since it was observed that equine lactating mammary gland contains significantly more mammosphere-initiating cells than the inactive, nonlactating gland (a reflection of MaSC self-renewal) and, moreover, that these spheres were significantly larger in size upon initial cultivation (a reflection of progenitor cell proliferation). Taken together, this study not only extends the current knowledge of mammary gland biology, but also benefits the comparative approach to study and compare MaSC in different mammalian species.

  7. Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines.

    PubMed

    Michishita, M; Akiyoshi, R; Suemizu, H; Nakagawa, T; Sasaki, N; Takemitsu, H; Arai, T; Takahashi, K

    2012-08-01

    Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

  8. Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and Epithelial Factors.

    PubMed

    Tobar, Nicolas; Porras, Omar; Smith, Patricio C; Barros, L Felipe; Martínez, Jorge

    2017-01-01

    Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc.

  9. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1

    SciTech Connect

    Desprez, P.; Hara, E.; Bissell, M.J.

    1995-06-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

  10. Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype.

    PubMed

    Cravero, Diego; Martignani, Eugenio; Miretti, Silvia; Accornero, Paolo; Baratta, Mario

    2015-10-01

    We previously proved that adult stem cells reside in the bovine mammary gland and possess an intrinsic potential to generate a functional mammary outgrowth. The aim of this study was to investigate on the immunophenotyping features retained by mammary stem-like cells detected in long term culture. Flow cytometry analysis showed different subpopulations of mammary epithelial cells emerging according to the timing of cell culture. CD49f(+)-cells significantly increased during the culture (p<0.01) and a similar trend was observed, even if less regular, for CD29(+) and ALDH1 positive cell populations. No difference during the culture was observed for CD24 positive cells but after 35 days of culture a subset of cells, CD49f positive, still retained regenerative capabilities in in vivo xenotransplants. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice. These results prove the presence of a multipotent cell subpopulation that retain a strong epithelial induction, confirmed in in vivo xenotransplants with a presumable in vitro expansion of the primitive population of adult mammary stem cells.

  11. An evaluation of the putative human mammary tumour retrovirus associated with peripheral blood monocytes.

    PubMed Central

    Kahl, L. P.; Carroll, A. R.; Rhodes, P.; Wood, J.; Read, N. G.

    1991-01-01

    The primary aims of this study were purification and molecular cloning of a putative retrovirus designated human mammary tumour virus (HMTV). However, our preliminary unpublished data of negative reverse transcriptase (RT) activity in ostensibly 'infected' cells led us to re-examine the evidence for this virus; namely multinucleate giant cell (MNGC) formation and RT activity in cultured blood monocytes from breast cancer patients versus benign breast tumour and normal control subjects. MNGCs from by fusion of monocytes and we estimated the total number of cell fusions which had occurred after 10 days of culture in vitro by counting cells with two, three, four and five or more nuclei (n) and by measuring the density of adherent mononuclear cells for each subject studied. We found no clear-cut difference in MNGC formation between the three subject groups. Moreover, a substantial number of cultures, encompassing the three groups, showed far more MNGCs per 10(5) monocytes than previously reported. Various parametric and nonparametric statistical analyses were performed on the multinucleate cell data and only one parametric test, which utilised the density of monolayers as a co-variate, showed a statistically significant difference at the 5% level between the breast cancer and the normal subject groups. We observed marked subject-to-subject variation in multinucleate cell formation and we suggest that the evidence for a difference between the breast cancer and the normal groups is marginal. Further, MNGC formation by breast cancer monocytes may not be attributed to the presence of a retrovirus since 5'-Azacytidine (AZA), an agent known to stimulate replication of latent retroviruses showed no effect on the MNGC formation. In addition, culture supernatants from the three groups were assayed for RT activity and no test sample gave a significant signal above background. Preliminary transmission electron microscopy analysis failed to identify viral particles in MNGCs

  12. Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells.

    PubMed

    Hasumi, K; Otsuki, R; Endo, A

    1985-08-01

    Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.

  13. Transplantation of a cell line derived from a canine benign mixed mammary tumour into nude mice.

    PubMed

    Priosoeryanto, B P; Tateyama, S; Yamaguchi, R; Uchida, K

    1995-11-01

    The MCM-B2 canine mammary cell line was serially transplanted into nude mice. The tumour masses consisted of elongated pleomorphic cells of varying size in the first to third passages; oval cells, becoming rounder, in the sixth to eighth passages; and cord-like, glandular and duct-like structures with compact radiating projections in the ninth and tenth passages. Ultrastructural and immunohistochemical examination of round cells confirmed their epithelial cell nature, but the morphology of the elongated and oval cells was identical with that of the original cell line. The findings suggest that the MCM-B2 cell line is a multipotential stem cell or is derived from glandular differentiation of mammary gland.

  14. Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation

    PubMed Central

    Binder, April K.; Kosak, Justin P.; Janhardhan, Kyathanahalli S.; Moser, Glenda; Eling, Thomas E.; Korach, Kenneth S.

    2016-01-01

    Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to

  15. Mammary Stem/Progenitor Cells and Cancer Susceptibility

    DTIC Science & Technology

    2012-06-01

    dependent mammary cancer; and 2) Brown Norway (BN), which is highly resistant. Major findings: ACI and BN rats likely exhibit significant differences in...stimulated states in two well characterized inbred rat strains: 1) ACI, which is highly susceptible to 17β-estradiol (E2)-induced/progesterone (P...MaSC number and/or responsiveness to hormone. Technical issues and loss of commercial source of BN rats led to revision of Aims. The data generated

  16. Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours

    PubMed Central

    Tao, Luwei; Xiang, Dongxi; Xie, Ying; Bronson, Roderick T.; Li, Zhe

    2017-01-01

    Most breast cancers may have a luminal origin. TP53 is one of the most frequently mutated genes in breast cancers. However, how p53 deficiency contributes to breast tumorigenesis from luminal cells remains elusive. Here we report that induced p53 loss in Krt8+ mammary luminal cells leads to their clonal expansion without directly affecting their luminal identity. All induced mice develop mammary tumours with 9qA1 (Yap1) and/or 6qA2 (Met) amplification(s). These tumours exhibit a mammary stem cell (MaSC)-like expression signature and most closely resemble claudin-low breast cancer. Thus, although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumours with loss of luminal identity. Our data also suggest that claudin-low breast cancer can develop from luminal cells, possibly via a basal-like intermediate state, although further study using a different luminal promoter is needed to fully support this conclusion. PMID:28194015

  17. Influence of selenium on the growth of N-nitrosomethylurea-induced mammary tumor cells in culture

    SciTech Connect

    Lewko, W.M.; McConnell, K.P.

    1985-10-01

    Selenium is an essential dietary trace element which has anticancer properties. Among its effects in rats, selenium has been shown to inhibit the development of carcinogen-induced mammary tumors by interfering with the post-initiation, promotion phase of carcinogenesis. We studied the effects of selenium on the growth of rat mammary tumor cells in primary culture. The objective was to determine whether selenium had any direct influence on cell growth which might explain its influence on tumor development. Rat mammary tumors were induced by N-nitrosomethylurea. The addition of low concentrations of sodium selenite, less than 1.0 ..mu..g/ml, stimulated tumor cell proliferation. Protein synthesis and the production of type IV collagen increased within the first hour of exposure, prior to any measurable increase in DNA synthesis. Concentrations of selenite greater than 1.0 ..mu..g/ml inhibited cell proliferation, the synthesis of protein, and the replication of DNA in a dose-related manner. These studies demonstrated that selenium has the potential to influence the post-initiation phase of rat mammary tumorigenesis by directly altering the growth of tumor cells, possibly through the regulation of protein synthesis.

  18. Genistein-mediated inhibition of mammary stromal adipocyte differentiation limits expansion of mammary stem/progenitor cells by paracrine signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammary adiposity may contribute to breast cancer development and progression by releasing cytokines and other inflammatory mediators that promote mammary epithelial proliferation. We evaluated the effects of soy isoflavone genistein (GEN) on the adipogenic differentiation of a SV40-immortalized mou...

  19. 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.

  20. Regulated expression of mouse mammary tumor proviral genes in cells of the B lineage

    PubMed Central

    1991-01-01

    We evaluated the expression of mouse mammary tumor proviral (MMTV) transcripts during B cell ontogeny and compared levels of RNA in B lymphocytes and B cell lines with levels in other cells of the hematopoietic lineage and in a mammary cell line. We demonstrate that MMTV transcripts are expressed as early as the pro-B cell stage in ontogeny and are expressed at basal constitutive levels throughout most of the B cell developmental pathway. The level of MMTV expression in B cells is similar to constitutive levels in mammary tissues and two to three orders of magnitude greater than in activated T cells. Levels of MMTV transcripts in B cells are not solely due to positional effects. Transient transfection assays showed that MMTV upregulation resulted from transcriptional activation of the viral LTR, indicating that there are specific and inducible transcription factors that regulate MMTV expression in B cells. MMTV transcripts could not be upregulated in pre- B cell lines but could be induced in some mature B cell lines. There was a correlation between the ability to stimulate B cells to secrete antibody and the ability to induce upregulated MMTV expression. Evidence is presented that suggests that the principal transcription factors involved in MMTV expression do not include the B cell factors OTF-2 or NF-kappa B, but rather are likely to be novel factors that are induced during differentiation to antibody secretion. A hypothesis for why mammary tumor viruses are well adapted for expression in cells of the B lineage is proposed, and the implications of this for the documented influence of MMTV gene products on the T cell repertoire are discussed. PMID:1660524

  1. Developing a Novel Mouse Model for Breast Cancer by Targeting Oncogenes to Mammary Progenitor Cells

    DTIC Science & Technology

    2005-04-01

    obtained and rederived into our facility this line of mice from another investigator Tom O’Brien (1). We are in the process of testing this line for...Breed K6-rtTA transgenic mice with established tetO-c-Myc mice. Transplant the mammary cells from the resulting bi -transgenic mice into 10 inguinal...concentrated RCAS-K-RasG12D-IRES-Cre virus to inguinal mammary glands of 10 pubertal and 10 estrogen-stimulated K6-TVA /p53flox/flox bi -genic females. Monitor

  2. Role of p53 in Mammary Epithelial Cell Senescence

    DTIC Science & Technology

    2007-05-01

    Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins . Annu Rev Genet 2004;38:413–43. 2... memory by the Polycomb and Trithorax group proteins . Annu. Rev. Genet. 38, 413–443. Rosenthal, N. (2005). Youthful prospects for human stem-cell...Javits Convention Center, New York City, NY 11 2007: “ Regulation of cell proliferation and senescence by Polycomb group

  3. Simvastatin exhibits antiproliferative effects on spheres derived from canine mammary carcinoma cells.

    PubMed

    Torres, Cristian G; Olivares, Araceli; Stoore, Caroll

    2015-05-01

    Mammary cancer is the most frequent type of tumor in the female canine. Treatments are mainly limited to surgery and chemotherapy; however, these tumors may develop clinical recurrence, metastasis and chemoresistance. The existence of a subpopulation of cancer cells with stemness features called cancer stem-like cells, may explain in part these characteristics of tumor progression. The statins, potent blockers of cholesterol synthesis, have also shown antitumor effects on cancer mammary cells, changes mediated by a decrease in the isoprenylation of specific proteins. Few studies have shown that simvastatin, a lipophilic statin, sensitizes cancer stem-like cells eliminating drug resistance. The aim of the present study was to evaluate the effects of simvastatin on spheres derived from CF41.Mg canine mammary tumor cells, which were characterized by phenotypic and functional analyses. Spheres exhibited characteristics of stemness, primarily expressing a CD44⁺/CD24⁻/low phenotype, displaying auto-renewal and relative chemoresistance. Exposure to simvastatin induced a decrease in the sphere-forming capacity and cell viability, accompanied by a concentration- and time-dependent increase in caspase-3/7 activity. In addition, modulation of β-catenin and p53 expression was observed. Simvastatin triggered a synergistic effect with doxorubicin, sensitizing the spheres to the cytotoxic effect exerted by the drug. Invasiveness of spheres was decreased in response to simvastatin and this effect was counteracted by the presence of geranylgeranyl pyrophosphate. Our results suggest that simvastatin targets canine mammary cancer stem-like cells, supporting its therapeutical application as a novel agent to treat canine mammary cancer.

  4. Identification of six potential markers for the detection of circulating canine mammary tumour cells in the peripheral blood identified by microarray analysis.

    PubMed

    da Costa, A; Lenze, D; Hummel, M; Kohn, B; Gruber, A D; Klopfleisch, R

    2012-01-01

    The presence of circulating tumour cells (CTCs) in the peripheral blood is a prognostic factor for survival of human breast cancer patients. CTCs in the peripheral blood of dogs with mammary tumours have not been reported definitively. The present pilot study identifies mRNA markers for CTCs by comparing the transcriptome of canine mammary carcinoma cell lines CMM26 and CMM115 and peripheral blood leucocytes (PBLs). Genes with a 200-fold or higher mRNA expression in carcinoma cell lines were tested for specificity and sensitivity to detect CTCs using reverse transcriptase polymerase chain reaction (PCR). Six mRNA markers, AGR2, ATP8B1, CRYAB, F3 IRX3 and SLC1A1 were expressed in cell lines, but not PBL. All PCRs were able to detect one carcinoma cell admixed in 10(6) or more PBLs. The six mRNA markers may be suitable for detection of canine mammary CTCs and allow the analysis of their spatiotemporal distribution in dogs with mammary tumours.

  5. 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

  6. The hedgehog signaling network, mammary stem cells, and breast cancer: connections and controversies.

    PubMed

    Lewis, M T; Visbal, A P

    2006-01-01

    Several signal transduction networks have been implicated in the regulation of mammary epithelial stem cell self-renewal and maintenance (Kalirai and Clarke 2006; Liu et al. 2005). These signaling networks include those of the Wnt, Notch, TGFO, EGF, FGF, IGF, and most recently, the Hedgehog (Hh) families of secreted ligands. However, we currently know very little about the cellular and molecular mechanisms by which these signaling pathways function to regulate normal epithelial stem/progenitor cells. What is clear is that the regulatory signaling networks thought to control normal stem/progenitor cell self-renewal and maintenance are, with the current sole exception of the hedgehog network, well-documented to have contributory roles in mammary cancer development and disease progression when misregulated. In this review, genetic regulation of mammary gland development by hedgehog network genes is outlined, highlighting a developing controversy as to whether activated hedgehog signaling regulates normal regenerative mammary epithelial stem cells or, indeed, whether activated hedgehog signaling functions at all in ductal development. In addition, the question of whether inappropriate hedgehog network activation influences breast cancer development is addressed, with emphasis on the prospects for using hedgehog signaling antagonists clinically for breast cancer treatment or prevention.

  7. Effect of elevated selenium intakes on mammary cell proliferation in rats

    SciTech Connect

    Salbe, A.D.; Albanes, D.; Winick, M.; Taylor, P.R.; Levander, O.A. National Institutes of Health, Bethesda, MD )

    1991-03-15

    Elevated selenium (Se) intakes and calorie restriction (CR) inhibit mammary tumorigenesis in experimental animals. The present study was designed to investigate cell proliferation in the mammary tissue gland. Female weanling Sprague-Dawley rats were divided into 4 groups: control, 40% CR, 4 or 6 ppm Se in water as selenate. Control rats and Se rats were fed a control diet ad lib. CR rats were pair-fed 40% less than controls with a diet providing equal nutrients except carbohydrate calories. After 3 weeks, rats were injected with ({sup 3}H)-thymidine and killed 1 hr later. Se at 4 ppm significantly decreased only the number of ducts, whereas 6 ppm Se decreased both the number of ducts as well as the number and percent of labeled cells. CR had no effect on mammary cell proliferation. These results suggest that elevated Se intakes may protect against mammary tumorigenesis by decreasing cell proliferation, a mechanism which may affect the dose-response of the genotoxic chemicals frequently used as initiating agents in animal experiments.

  8. Myoepithelial cell differentiation markers in prepubertal bovine mammary gland: Effect of ovariectomy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously reported that ovariectomy alters prepubertal development of mammary myoepithelial cells (MC), but the mechanisms involved are not well understood. We therefore analyzed the expression of the myoepithelial differentiation markers a-smooth muscle actin (SMA) and the common acute ly...

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

    SciTech Connect

    Simian, Marina; Hirai, Yohei; Navre, Marc; Werb, Zena; Lochter, Andre; Bissell, Mina J.

    2002-03-06

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

  10. Examination of Duct Physiology in the Human Mammary Gland

    PubMed Central

    Mills, Dixie; Gomberawalla, Ameer; Gordon, Eva J.; Tondre, Julie; Nejad, Mitra; Nguyen, Tinh; Pogoda, Janice M.; Rao, Jianyu; Chatterton, Robert; Henning, Susanne; Love, Susan M.

    2016-01-01

    Background The human breast comprise several ductal systems, or lobes, which contain a small amount of fluid containing cells, hormones, proteins and metabolites. The complex physiology of these ducts is likely a contributing factor to the development of breast cancer, especially given that the vast majority of breast cancers begin in a single lobular unit. Methods We examined the levels of total protein, progesterone, estradiol, estrone sulfate, dehydroepiandrosterone sulfate, and macrophages in ductal fluid samples obtained from 3 ducts each in 78 women, sampled twice over a 6 month period. Samples were processed for both cytological and molecular analysis. Intraclass correlation coefficients and mixed models were utilized to identify significant data. Results We found that the levels of these ductal fluid components were generally uncorrelated among ducts within a single breast and over time, suggesting that each lobe within the breast has a distinct physiology. However, we also found that estradiol was more correlated in women who were nulliparous or produced nipple aspirate fluid. Conclusions Our results provide evidence that the microenvironment of any given lobular unit is unique to that individual unit, findings that may provide clues about the initiation and development of ductal carcinomas. PMID:27073976

  11. Comparative value of clinical, cytological, and histopathological features in feline mammary gland tumors; an experimental model for the study of human breast cancer

    PubMed Central

    2013-01-01

    Background The diagnosis of breast lesions is usually confirmed by fine-needle aspiration cytology (FNAC) or histological biopsy. Although there is increasing literature regarding the advantages and limitations of both modalities, there is no literature regarding the accuracy of these modalities for diagnosing breast lesions in high-risk patients, who usually have lesions detected by screening. Moreover, few studies have been published regarding the cytopathology of mammary tumors in cats despite widespread use of the animal model for breast cancer formation and inhibition. The objective of the present study was to evaluate the diagnostic interest of cytological and histopathological analysis in feline mammary tumours (FMTs), in order to evaluate its possible value as an animal model. Methods The study was performed in 3 female cats submitted to surgical resections of mammary tumours. The mammary tumours were excised by simple mastectomy or regional mastectomy, with or without the superficial inguinal lymph nodes. Female cats were of different breeds (1 siamese and 2 persians). Before surgical excision of the tumour, FNA cytology was performed using a 0.4 mm diameter needle attached to a 8 ml syringe held in a standard metal syringe holder. The cytological sample was smeared onto a glass slide and either air-dried for May-Grünwald-stain and masses were surgically removed, the tumours were grossly examined and tissue samples were fixed in 10%-buffered-formalin and embedded in paraffin. Sections 4 μm thick were obtained from each sample and H&E stained. Results Cytologically, atypical epithelial cells coupled to giant nucleus, chromatin anomalies, mitotic figures, spindle shape cells, anisocytosis with anisokaryosis and hyperchromasia were found. Histologically, these tumors are characterized by pleomorphic and polygonal cell population together with mitotic figures, necrotic foci and various numbers inflammatory foci. Also, spindle shaped cells, haemorrhage

  12. Developmental signaling pathways regulating mammary stem cells and contributing to the etiology of triple-negative breast cancer.

    PubMed

    Rangel, Maria Cristina; Bertolette, Daniel; Castro, Nadia P; Klauzinska, Malgorzata; Cuttitta, Frank; Salomon, David S

    2016-04-01

    Cancer has been considered as temporal and spatial aberrations of normal development in tissues. Similarities between mammary embryonic development and cell transformation suggest that the underlying processes required for mammary gland development are also those perturbed during various stages of mammary tumorigenesis and breast cancer (BC) development. The master regulators of embryonic development Cripto-1, Notch/CSL, and Wnt/β-catenin play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells (fMaSC) and in the adult organism particularly within the adult mammary stem cells (aMaSC), which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during tumorigenesis to support the tumor-initiating cell (TIC)/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of BC, in particular the most aggressive subtype of BC, triple-negative breast cancer (TNBC). This in depth review will present evidence of the involvement of Cripto-1, Notch/CSL, and Wnt/β-catenin in the normal mammary gland morphogenesis and tumorigenesis, from fMaSC/aMaSC regulation to TIC generation and maintenance in TNBC. Specific therapies for treating TNBC by targeting these embryonic pathways in TICs will be further discussed, providing new opportunities to destroy not only the bulk tumor, but also TICs that initiate and promote the metastatic spread and recurrence of this aggressive subtype of BC.

  13. Models of breast morphogenesis based on localization of stem cells in the developing mammary lobule.

    PubMed

    Honeth, Gabriella; Schiavinotto, Tommaso; Vaggi, Federico; Marlow, Rebecca; Kanno, Tokuwa; Shinomiya, Ireneusz; Lombardi, Sara; Buchupalli, Bharath; Graham, Rosalind; Gazinska, Patrycja; Ramalingam, Vernie; Burchell, Joy; Purushotham, Anand D; Pinder, Sarah E; Csikasz-Nagy, Attila; Dontu, Gabriela

    2015-04-14

    Characterization of normal breast stem cells is important for understanding their role in breast development and in breast cancer. However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known. In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals. Comparing predictions of these models with immunohistochemical analysis of tissue sections for candidate stem cell markers, we defined distinct areas where stem cells reside in the mammary lobule. An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of nulliparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

  14. Breast Cancer and Early Onset Childhood Obesity: Cell Specific Gene Expression in Mammary Epithelia and Adipocytes

    DTIC Science & Technology

    2007-07-01

    hormone leptin (ob/ob mice) or its receptor (db/db mice, Zucker rat). These leptin signaling impaired animals are resistant to oncogene and...chemically induced mammary tumors (3,4). However, human obesity is not generally caused by mutations in leptin or its receptor (5). As expression of leptin ...morbidity factors associated with human obesity in the three groups of rats, including Leptin , Free fatty acids (FFA), triglycerides (TG) and insulin

  15. STAT5A Regulates the Survival of Mammary Epithelial Cells and the Development of Mammary Cancer

    DTIC Science & Technology

    2000-07-01

    Bill Kemp, and Sandra Price for animal husbandry for 5 h in Tellyzinckys fixative, and stored in 70% ethanol until processed and technical support and...of tumor formation was the same as the control group. This suggested that the process of involution can remove significant numbers of cells that could...potentially contribute to the development of tumors. But it also suggested that there remained sufficient cells after the process of involution, that

  16. Antiproliferative effect of berberine on canine mammary gland cancer cell culture.

    PubMed

    Sefidabi, Reyhaneh; Mortazavi, Pejman; Hosseini, Saeed

    2017-01-01

    Canine mammary gland tumors are the most frequent cause of cancer in female dogs. Numerous studies using cancer cell lines and clinical trials have indicated that various natural products and antioxidants reduce or possibly prevent the development of cancer. Berberine (BBR), the most important alkaloid in the Berberidaceae, which exerts a wide range of pharmacological and biochemical effects, has drawn much attention due to its particularly high antitumor activity in vitro and in animal studies. The aim of the present study was to investigate the antiproliferative effect of BBR against a canine mammary gland carcinoma cell line (CF41.Mg) in vitro. CF41.Mg cells were cultured in RPMI-1640 medium containing 10% heat inactived fetal bovine serum (FBS) and 100 mg/ml peniciline-streptomycin. Subsequently the cells were treated with different concentrations of BBR chloride (10, 25, 50, 100 and 200 µM) at a density of 12,000 cells/well in 96-well plates. Following treatment, the MTT assay was used to detect cell viability after 24-, 48- and 72-h incubations at 37°C with 5% CO2. The results indicated that BBR inhibited proliferation of canine mammary gland carcinoma cells, as treatment with 100 µM BBR for 24 h resulted in a significant decrease in cell viability (P<0.005). As the present study demonstrated that BBR (10-200 µM) induced cancer cell death, it is proposed that BBR may serve as a candidate agent against canine mammary tumor cells via its antiproliferative activity.

  17. Canine mammary tumors contain cancer stem-like cells and form spheroids with an embryonic stem cell signature.

    PubMed

    Ferletta, Maria; Grawé, Jan; Hellmén, Eva

    2011-01-01

    We have investigated the presence of tentative stem-like cells in the canine mammary tumor cell line CMT-U229. This cell line is established from an atypical benign mixed mammary tumor, which has the property of forming duct-like structures in collagen gels. Stem cells in mammary glands are located in the epithelium; therefore we thought that the CMT-U229 cell line would be suitable for detection of tentative cancer stem-like cells. Side population (SP) analyses by flow cytometry were performed with cells that formed spheroids and with cells that did not. Flow cytometric, single sorted cells were expanded and re-cultured as spheroids. The spheroids were paraffin embedded and characterized by immunohistochemistry. SP analyses showed that spheroid forming cells (retenate) as well as single cells (filtrate) contained SP cells. Sca1 positive cells were single cell sorted and thereafter the SP population increased with repeated SP analyses. The SP cells were positively labeled with the cell surface-markers CD44 and CD49f (integrin alpha6); however the expression of CD24 was low or negative. The spheroids expressed the transcription factor and stem cell marker Sox2, as well as Oct4. Interestingly, only peripheral cells of the spheroids and single cells were positive for Oct4 expression. SP cells are suggested to correspond to stem cells and in this study, we have enriched for tentative tumor stem-like cells derived from a canine mammary tumor. All the used markers indicate that the studied CMT-U229 cell line contains SP cells, which in particular have cancer stem-like cell characteristics.

  18. Non-autonomous cell proliferation in the mammary gland and cancer.

    PubMed

    Weber, Robert J; Desai, Tejal A; Gartner, Zev J

    2017-03-14

    Cells decide whether to grow and divide by integrating internal and external signals. Non-autonomous cell growth and proliferation occurs when microenvironmental signals from neighboring cells, both physical and secreted, license this decision. Understanding these processes is vital to developing an accurate framework for cell-cell interactions and cellular decision-making, and is useful for advancing new therapeutic strategies to prevent dysregulated growth. Here, we review some recent examples of non-autonomous cell growth in the mammary gland and tumor cell proliferation.

  19. Mammary epithelial cell phenotype disruption in vitro and in vivo through ERalpha36 overexpression

    PubMed Central

    Chesnel, Amand; Morel, Chloé; Djermoune, El-Hadi; Boukhobza, Taha

    2017-01-01

    Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), widely expressed in hormone sensitive cancer cells and whose high expression level correlates with a poor survival prognosis for breast cancer patients. While ERα36 activity have been related to breast cancer progression or acquired resistance to treatment, expression level and location of ERα36 are poorly documented in the normal mammary gland. Therefore, we explored the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein ERα36 mRNA was specifically expressed in the mammary gland. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36 and modulated by the JAK2/STAT3 signaling pathway. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration potential and resistance to staurosporin-induced apoptosis of the MCF-10A cell line. In vivo, ERα36 expression led to duct epithelium thinning and disruption in adult but not in prepubescent mouse mammary gland. These phenotypes correlated with a loss of E-cadherin expression. Here, we show that an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of adult endogenous steroid levels, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk. PMID:28301550

  20. Establishment and characterization of a new human oestradiol- and progesterone-receptor-positive mammary carcinoma serially transplantable in nude mice.

    PubMed

    Naundorf, H; Fichtner, I; Büttner, B; Frege, J

    1992-01-01

    A human mammary carcinoma originating from a postmenopausal patient was successfully transplanted into nude mice. According to the adopted criteria the tumour proved to be oestradiol- and progesterone-receptor-positive. Histological studies of the patient tumour revealed a ductal invasive mammary carcinoma with 80% tubular growth pattern. Following transplantation the adenoid structures decreased to 30%; the mitosis rate and grade of malignancy increased. Treatment of the nude mice with 20 micrograms oestradiol benzoate/mouse caused a loss of the oestradiol receptor of the mammary carcinoma. The mammary carcinoma 3366 can be used for testing of antineoplastic substances, antihormones and for studies in regard to down-regulation or blocking of hormone receptors and possible consequences for therapies.

  1. 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.

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

    PubMed

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

    2013-02-08

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

  3. An essential role for Gα(i2) in Smoothened-stimulated epithelial cell proliferation in the mammary gland.

    PubMed

    Villanueva, Hugo; Visbal, Adriana P; Obeid, Nadine F; Ta, Andrew Q; Faruki, Adeel A; Wu, Meng-Fen; Hilsenbeck, Susan G; Shaw, Chad A; Yu, Peng; Plummer, Nicholas W; Birnbaumer, Lutz; Lewis, Michael T

    2015-09-15

    Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. The gene encoding Smoothened (SMO), the primary effector of Hh signaling, is expressed aberrantly in human breast cancer, as well as in other cancers. In mice that express a constitutively active form of SMO that does not require Hh stimulation in mammary glands, the cells near the transgenic cells proliferate and participate in hyperplasia formation. Although SMO is a seven-transmembrane receptor like G protein-coupled receptors (GPCRs), SMO-mediated activation of the Gli family of transcription factors is not known to involve G proteins. However, data from Drosophila and mammalian cell lines indicate that SMO functions as a GPCR that couples to heterotrimeric G proteins of the pertussis toxin (PTX)-sensitive Gαi class. Using genetically modified mice, we demonstrated that SMO signaling through G proteins occurred in the mammary gland in vivo. SMO-induced stimulation of proliferation was PTX-sensitive and required Gαi2, but not Gαi1, Gαi3, or activation of Gli1 or Gli2. Our findings show that activated SMO functions as a GPCR to stimulate proliferation in vivo, a finding that may have clinical importance because most SMO-targeted agents have been selected based largely on their ability to block Gli-mediated transcription.

  4. The myoepithelial cell: its role in normal mammary glands and breast cancer.

    PubMed

    Sopel, M

    2010-02-01

    Mammary gland epithelium is composed of an inner layer of secretory cells (luminal) and an outer layer of myoepithelial cells (MEC) bordering the basal lamina which separates the epithelial layer from the extracellular matrix. Mature MECs morphologically resemble smooth muscle cells; however, they exhibit features typical for epithelial cells, such as the presence of specific cytokeratin filaments. During lactation, secretory cells synthesize milk components, which are collected in alveoli and duct lumen, and transported to the nipple as a result of MEC contraction. Although the induction of MEC contraction results from oxytocin action, also other, still unknown auto/paracrine mechanisms participate in the regulation of this process. As well as milk ejection, MECs are involved in mammary gland morphogenesis in all developmental stages, modulating proliferation and differentiation of luminal cells. They take part in the formation of extracellular matrix, synthesizing its components and secreting proteinases and their inhibitors. In addition, MECs are regarded as natural cancer suppressors, stabilizing the normal structure of the mammary gland, they secrete suppressor proteins (e.g. maspin) limiting cancer growth, invasiveness, and neoangiogenesis. The majority of malignant breast cancers are derived from luminal cells, whereas neoplasms of MEC origin are the most seldom and usually benign form of breast tumours. MECs are markedly resistant to malignant transformation and they are able to suppress the transformation of neighboring luminal cells. Therefore, a deeper insight into the role of MECs in the physiology and pathology of mammary glands would allow a better understanding of cancerogenesis mechanisms and possible application of specific MEC markers in the diagnosis and therapy of breast cancer.

  5. STAT5A Regulates the Survival of Mammary Epithelial Cells and the Development of Mammary Cancer

    DTIC Science & Technology

    2002-07-01

    cleared. Gel was dried according to the man- stored in 7 0% ethanol until processed by standard embedding and ufacturer’s protocol (Research Products...fixative, and stored in 70% ethanol until processed and technical support and Dr. J. Shillingford for developing the Stat5a by standard embedding and...that the process of involution could remove significant numbers of cells that could potentially contribute to the development of tumors. But it also

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    SciTech Connect

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

    2002-02-22

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

  9. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

    PubMed

    Lin, Weiming; Modiano, Jaime F; Ito, Daisuke

    2017-03-30

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1(-) and SSEA-1(+) cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

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

    SciTech Connect

    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. - 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.

  11. Clumping factor A of Staphylococcus aureus interacts with AnnexinA2 on mammary epithelial cells

    PubMed Central

    Ashraf, Shoaib; Cheng, Jing; Zhao, Xin

    2017-01-01

    Staphylococcus aureus is one of major pathogens that can cause a series of diseases in different hosts. In the bovine, it mainly causes subclinical and contagious mastitis, but its mechanisms of infection are not fully understood. Considering the fact that virulence factors play key roles in interactions between the bacterium and host cells, this study aimed to identify if a binding partner of S. aureus clumping factor A (ClfA) exists on the bovine mammary epithelial cells. The ClfA protein was used as a bait to pull down lysates of cultured bovine mammary epithelial cells (MAC-T cells). One pull-down protein was identified through use of mass spectrometry and bioinformatics analyses as bovine AnnexinA2. The Western blot and in vitro binding assay confirmed that the full A domain of ClfA was necessary to bind to AnnexinA2. In addition, the interaction between ClfA and AnnexinA2 was validated biochemically by ELISA with a KD value of 418+/−93 nM. The confocal microscopy demonstrated that ClfA and AnnexinA2 partially co-localized in the plasma membrane and that the majority of them were transported into cytoplasm. Taken together, the results demonstrate that ClfA binds with AnnexinA2 and this interaction could mediate S. aureus invasion into bovine mammary epithelial cells. PMID:28102235

  12. Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells

    PubMed Central

    Tavera-Mendoza, Luz E.; Westerling, Thomas; Libby, Eric; Marusyk, Andriy; Cato, Laura; Cassani, Raymundo; Cameron, Lisa A.; Ficarro, Scott B.; Marto, Jarrod A.; Klawitter, Jelena; Brown, Myles

    2017-01-01

    Women in North America have a one in eight lifetime risk of developing breast cancer (BC), and a significant proportion of these individuals will develop recurrent BC and will eventually succumb to the disease. Metastatic, therapy-resistant BC cells are refractory to cell death induced by multiple stresses. Here, we document that the vitamin D receptor (VDR) acts as a master transcriptional regulator of autophagy. Activation of the VDR by vitamin D induces autophagy and an autophagic transcriptional signature in BC cells that correlates with increased survival in patients; strikingly, this signature is present in the normal mammary gland and is progressively lost in patients with metastatic BC. A number of epidemiological studies have shown that sufficient vitamin D serum levels might be protective against BC. We observed that dietary vitamin D supplementation in mice increases basal levels of autophagy in the normal mammary gland, highlighting the potential of vitamin D as a cancer-preventive agent. These findings point to a role of vitamin D and the VDR in modulating autophagy and cell death in both the normal mammary gland and BC cells. PMID:28242709

  13. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  14. Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells.

    PubMed

    Tavera-Mendoza, Luz E; Westerling, Thomas; Libby, Eric; Marusyk, Andriy; Cato, Laura; Cassani, Raymundo; Cameron, Lisa A; Ficarro, Scott B; Marto, Jarrod A; Klawitter, Jelena; Brown, Myles

    2017-03-14

    Women in North America have a one in eight lifetime risk of developing breast cancer (BC), and a significant proportion of these individuals will develop recurrent BC and will eventually succumb to the disease. Metastatic, therapy-resistant BC cells are refractory to cell death induced by multiple stresses. Here, we document that the vitamin D receptor (VDR) acts as a master transcriptional regulator of autophagy. Activation of the VDR by vitamin D induces autophagy and an autophagic transcriptional signature in BC cells that correlates with increased survival in patients; strikingly, this signature is present in the normal mammary gland and is progressively lost in patients with metastatic BC. A number of epidemiological studies have shown that sufficient vitamin D serum levels might be protective against BC. We observed that dietary vitamin D supplementation in mice increases basal levels of autophagy in the normal mammary gland, highlighting the potential of vitamin D as a cancer-preventive agent. These findings point to a role of vitamin D and the VDR in modulating autophagy and cell death in both the normal mammary gland and BC cells.

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

    SciTech Connect

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

    2009-04-20

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

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

    PubMed

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

    2016-03-11

    Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.

  17. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny

    PubMed Central

    Davis, Felicity M.; Lloyd-Lewis, Bethan; Harris, Olivia B.; Kozar, Sarah; Winton, Douglas J.; Muresan, Leila; Watson, Christine J.

    2016-01-01

    The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium. PMID:27779190

  18. Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model.

    PubMed

    Xiao, Changting; Quinton, V Margaret; Cant, John P

    2004-04-01

    Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-d-[1-(3)H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37 degrees C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 +/- 5.7% and 67.4 +/- 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.

  19. 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

  20. An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance.

    PubMed

    Mohapatra, Bhopal; Zutshi, Neha; An, Wei; Goetz, Benjamin; Arya, Priyanka; Bielecki, Timothy A; Mustaq, Insha; Storck, Matthew D; Meza, Jane L; Band, Vimla; Band, Hamid

    2017-03-15

    The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifen-inducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cbl(flox/flox); Cbl-b(flox/flox); Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

  1. SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

    PubMed Central

    Barutcu, A. Rasim; Lajoie, Bryan R.; Fritz, Andrew J.; McCord, Rachel P.; Nickerson, Jeffrey A.; van Wijnen, Andre J.; Lian, Jane B.; Stein, Janet L.; Dekker, Job; Stein, Gary S.; Imbalzano, Anthony N.

    2016-01-01

    The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization. PMID:27435934

  2. microRNAs and EMT in mammary cells and breast cancer.

    PubMed

    Wright, Josephine A; Richer, Jennifer K; Goodall, Gregory J

    2010-06-01

    MicroRNAs are master regulators of gene expression in many biological and pathological processes, including mammary gland development and breast cancer. The differentiation program termed the epithelial to mesenchymal transition (EMT) involves changes in a number of microRNAs. Some of these microRNAs have been shown to control cellular plasticity through the suppression of EMT-inducers or to influence cellular phenotype through the suppression of genes involved in defining the epithelial and mesenchymal cell states. This has led to the suggestion that microRNAs maybe a novel therapeutic target for the treatment of breast cancer. In this review, we will discuss microRNAs that are involved in EMT in mammary cells and breast cancer.

  3. Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland.

    PubMed

    Gruber, A D; Schreur, K D; Ji, H L; Fuller, C M; Pauli, B U

    1999-06-01

    The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

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

    PubMed

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

    2016-01-01

    The mammary epithelial cells (MECs) of high-producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide- (H2O2-) induced OS in cultured bovine MECs (MAC-T). Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after H2O2 exposure. Resveratrol helped MAC-T cells to prevent H2O2-induced endoplasmic reticulum stress and mitochondria-related cell apoptosis. Moreover, resveratrol induced mRNA expression of multiple antioxidant defense genes in MAC-T cells under normal/oxidative conditions. Nuclear factor erythroid 2-related factor 2 (Nrf2) was required for the cytoprotective effects on MAC-T cells by resveratrol, as knockdown of Nrf2 significantly abolished resveratrol-induced cytoprotective effects against OS. In addition, by using selective inhibitors, we further confirmed that the induction of Nrf2 by resveratrol was mediated through the prolonged activation of PI3K/Akt and ERK/MAPK pathways but negatively regulated by p38/MAPK pathway. Overall, resveratrol has beneficial effects on bovine MECs redox balance and may be potentially used as a therapeutic medicine against oxidative insult in lactating animals.

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

    PubMed Central

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

    2016-01-01

    The mammary epithelial cells (MECs) of high-producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide- (H2O2-) induced OS in cultured bovine MECs (MAC-T). Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after H2O2 exposure. Resveratrol helped MAC-T cells to prevent H2O2-induced endoplasmic reticulum stress and mitochondria-related cell apoptosis. Moreover, resveratrol induced mRNA expression of multiple antioxidant defense genes in MAC-T cells under normal/oxidative conditions. Nuclear factor erythroid 2-related factor 2 (Nrf2) was required for the cytoprotective effects on MAC-T cells by resveratrol, as knockdown of Nrf2 significantly abolished resveratrol-induced cytoprotective effects against OS. In addition, by using selective inhibitors, we further confirmed that the induction of Nrf2 by resveratrol was mediated through the prolonged activation of PI3K/Akt and ERK/MAPK pathways but negatively regulated by p38/MAPK pathway. Overall, resveratrol has beneficial effects on bovine MECs redox balance and may be potentially used as a therapeutic medicine against oxidative insult in lactating animals. PMID:26962394

  6. Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells.

    PubMed

    Accornero, P; Martignani, E; Miretti, S; Starvaggi Cucuzza, L; Baratta, M

    2009-08-01

    The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.

  7. Regulation of Stearoyl Coenzyme A Desaturase 1 Gene Promoter in Bovine Mammary Cells.

    PubMed

    di Martino, O; Troiano, A; Addi, L; Guarino, A; Calabrò, S; Tudisco, R; Murru, N; Cutrignelli, M I; Infascelli, F; Calabrò, V

    2015-01-01

    Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Bovine mammary epithelial cells (BME-UV1), cultured in vitro, have been proposed as a model to reproduce the biology of the mammary gland. The present study was designed to investigate the responsiveness of bovine SCD1 promoter to serum, insulin, oleic acid, and NFY transcription factor in BME-UV1 cells. A luciferase-based reporter assay was used to monitor the transcriptional activity of the SCD1 promoter region in BME-UV1 cells treated or not with insulin and/or oleic acid. The level of endogenous SCD1 mRNA was evaluated by Real time PCR. Insulin (20 ng/mL) induced a 2.0 to 2.5-fold increase of SCD1 promoter activity. Additionally, the effect of insulin was inhibited by oleic acid, serum components, and NFY enforced expression. Serum and NFY showed no synergistic or additive effect on SCD1 promoter activity suggesting that they repress SCD1 transcription through the same responsive element.

  8. Embryonic stem cell gene expression signatures in the canine mammary tumor: a bioinformatics approach.

    PubMed

    Zamani-Ahmadmahmudi, Mohamad

    2016-08-01

    Canine breast cancer was considered as an ideal model of comparative oncology for the human breast cancer, as there is significant overlap between biological and clinical characteristics of the human and canine breast cancer. We attempt to clarify expression profile of the embryonic stem cell (ES) gene signatures in canine breast cancer. Using microarray datasets (GSE22516 and GSE20718), expression of the three major ES gene signatures (modules or gene-sets), including Myc, ESC-like, and PRC modules, was primarily analyzed through Gene-Set Enrichment Analysis (GSEA) method in tumor and healthy datasets. For confirmation of the primary results, an additional 13 ES gene-sets which were categorized into four groups including ES expressed (ES exp1 and ES exp2), NOS targets (Nanog targets, Oct4 targets, Sox2 targets, NOS targets, and NOS TFs), Polycomb targets (Suz12 targets, Eed targets, H3K27 bound, and PRC2 targets), and Myc targets (Myc targets1, and Myc targets2) were tested in the tumor and healthy datasets. Our results revealed that there is a valuable overlap between canine and human breast cancer ES gene-sets expression profile, where Myc and ESC-like modules were up-regulated and PRC module was down-regulated in metastatic canine mammary gland tumors. Further analysis of the secondary gene-sets indicated overexpression of the ES expressed, NOS targets (Nanog targets, Oct4 targets, Sox2 targets, and NOS targets), and Myc targets and underexpression of the Polycomb targets in metastatic canine breast cancer.

  9. Cytokine networks that mediate epithelial cell-macrophage crosstalk in the mammary gland: implications for development and cancer.

    PubMed

    Sun, Xuan; Ingman, Wendy V

    2014-07-01

    Dynamic interactions between the hormone responsive mammary gland epithelium and surrounding stromal macrophage populations are critical for normal development and function of the mammary gland. Macrophages are versatile cells capable of diverse roles in mammary gland development and maintenance of homeostasis, and their function is highly dependent on signals within the local cytokine microenvironment. The mammary epithelium secretes a number of cytokines, including colony stimulating factor 1 (CSF1), transforming growth factor beta 1 (TGFB1), and chemokine ligand 2 (CCL2) that affect the abundance, phenotype and function of macrophages. However, aberrations in these interactions have been found to increase the risk of tumour formation, and utilisation of stromal macrophage support by tumours can increase the invasive and metastatic potential of the cancer. Studies utilising genetically modified mouse models have shed light on the significance of epithelial cell-macrophage crosstalk, and the cytokines that mediate this communication, in mammary gland development and tumourigenesis. This article reviews the current status of our understanding of the roles of epithelial cell-derived cytokines in mammary gland development and cancer, with a focus on the crosstalk between epithelial cells and the local macrophage population.

  10. Identification and characterization of microRNA sequences from bovine mammary epithelial cells.

    PubMed

    Bu, D P; Nan, X M; Wang, F; Loor, J J; Wang, J Q

    2015-03-01

    The bovine mammary gland is composed of various cell types including bovine mammary epithelial cells (BMEC). The use of BMEC to uncover the microRNA (miRNA) profile would allow us to obtain a more specific profile of miRNA sequences that could be associated with lactation and avoid interference from other cell types. The objective of this study was to characterize the miRNA sequences expressed in isolated BMEC. The miRNA were identified by Solexa sequencing technology (Illumina Inc., San Diego, CA). Furthermore, novel miRNA were uncovered by stem-loop reverse transcription-PCR and sequencing of PCR products. To detect tissue specificity, expression of novel miRNA sequences was measured by stem-loop RT-PCR and sequencing of PCR products in mammary gland, liver, adipose, ileum, spleen and kidney tissue from 3 lactating Holstein cows (50±10 d postpartum). After bioinformatics analysis, 12,323,451 reads were obtained by Solexa sequencing, of which 11,979,706 were clean reads, matching the bovine genome. Among clean reads, 9,428,122 belonged to miRNA sequences. Further analysis revealed that the miRNA bta-mir-184 had the most abundant expression, and 388 loci possessed the typical stem-loop structures matching known miRNA hairpins. In total, 38 loci with novel hairpins were identified as novel miRNA and were numbered from bta-U1 to bta-U38. One novel miRNA (bta-U21) was specific to mammary gland. Seven novel miRNA, including bta-U21, had tissue-restricted distribution. Uncovering the specific roles of these novel miRNA during lactation appears warranted.

  11. Mouse mammary tumor virus-like nucleotide sequences in canine and feline mammary tumors.

    PubMed

    Hsu, Wei-Li; Lin, Hsing-Yi; Chiou, Shyan-Song; Chang, Chao-Chin; Wang, Szu-Pong; Lin, Kuan-Hsun; Chulakasian, Songkhla; Wong, Min-Liang; Chang, Shih-Chieh

    2010-12-01

    Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats.

  12. Mouse Mammary Tumor Virus-Like Nucleotide Sequences in Canine and Feline Mammary Tumors▿

    PubMed Central

    Hsu, Wei-Li; Lin, Hsing-Yi; Chiou, Shyan-Song; Chang, Chao-Chin; Wang, Szu-Pong; Lin, Kuan-Hsun; Chulakasian, Songkhla; Wong, Min-Liang; Chang, Shih-Chieh

    2010-01-01

    Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats. PMID:20881168

  13. Mechanisms of modulation of the Egr gene family in mammary epithelial cells of different species.

    PubMed

    Santino, P; Martignani, E; Miretti, S; Baratta, M; Accornero, P

    2017-01-22

    In the adult female, within the estrous cycle, the mammary gland undergoes multiple rounds of growth, with increased cellular proliferation, and involution, with increased apoptosis. The increase in proliferation is elicited by endocrine (Estrogen, Progesterone), as well as locally produced (epidermal growth factor, insulin-like growth factor, etc) growth factors. Among the genes that are modulated during cellular proliferation, immediate early genes play a fundamental role, being rapidly upregulated and then downregulated within the G0/G1 phase of the cell cycle, allowing the progression to the subsequent phases. Egrs (1-4) are immediate early genes that encode for transcription factors that promote, within different cell types and depending on the strength and duration of the stimuli, several different responses like mitogenesis, differentiation, apoptosis or even anti-apoptosis. In this work we have studied the mechanisms of modulation of the Egr family, in mammary epithelial cells of different origin (bovine, canine, feline, murine). Following stimulation with growth medium, Egr mRNA expression showed a strong upregulation reaching a peak at 45-60min, that rapidly declined. Among several cytokines, particularly important for mammary morphogenesis, that we have tested (EGF, IGF-I, insulin, estrogen, progesterone), only EGF upregulated Egrs to levels close to those elicited by growth medium. In order to understand how the Egr transcription factors were regulated, we have inhibited Erk 1/2 and PI3K, molecules that drive two major intracellular signaling pathways. Inhibition of the Erk 1/2 pathway totally abolished Egr upregulation mediated by growth medium or EGF. On the other hand, the PI3K-Akt pathway played a minor role on Egr levels, with a strong inhibitory effect on cat GH2 cells only, that could be ascribed to reduced Erk phosphorylation following PI3K inhibition. Finally we showed that addition of growth medium also upregulated that the mammary luminal

  14. Regulation of adipocyte lipid homeostasis by genistein alters mammary epithelial cell differentiation: a paracrine mechanism for mammary tumor protection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epidemiological and animal studies have shown a negative correlation between breast cancer incidence and intake of soy rich foods. Our laboratory has studied soy protein isolate (SPI), the primary component of soy infant formula, as a paradigm to evaluate diet as a risk factor in mammary cancer. We ...

  15. Extracellular vesicles from women with breast cancer promote an epithelial-mesenchymal transition-like process in mammary epithelial cells MCF10A.

    PubMed

    Galindo-Hernandez, Octavio; Gonzales-Vazquez, Cristina; Cortes-Reynosa, Pedro; Reyes-Uribe, Emmanuel; Chavez-Ocaña, Sonia; Reyes-Hernandez, Octavio; Sierra-Martinez, Mónica; Salazar, Eduardo Perez

    2015-12-01

    Extracellular vesicles (EVs) mediate many stages of tumor progression including angiogenesis, escape from immune surveillance, and extracellular matrix degradation. We studied whether EVs from plasma of women with breast cancer are able to induce an epithelial-mesenchymal transition (EMT) process in mammary epithelial cells MCF10A. Our findings demonstrate that EVs from plasma of breast cancer patients induce a downregulation of E-cadherin expression and an increase of vimentin and N-cadherin expression. Moreover, EVs induce migration and invasion, as well as an increase of NFκB-DNA binding activity and MMP-2 and MMP-9 secretions. In summary, our findings demonstrate, for the first time, that EVs from breast cancer patients induce an EMT-like process in human mammary non-tumorigenic epithelial cells MCF10A.

  16. 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

  17. Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion

    SciTech Connect

    Lee, E.Y.H.P.; Lee, W.H.; Kaetzel, C.S.; Parry, G.; Bissell, M.J.

    1985-03-01

    Mouse mammary epithelial cells (MMEC) secrete certain milk proteins only when cultured on floating collagen gels. The authors demonstrate that modulation of milk proteins by substrata is manifested at several regulatory levels; (i) cells cultured on floating collagen gels have 3- to 10-fold more casein mRNA than cells cultured on plastic or attached collagen gels. (ii) Cells on the latter two flat substrata, nevertheless, synthesize a significant amount of caseins, indicating that the remaining mRNA is functional. (iii) Cells on all substrata are inducible for casein mRNA and casein proteins by prolactin, but the extent of induction is greater on collagen than that on plastic - i.e., the substratum confers an altered degree of inducibility. (iv) Cells on all substrata synthesize casein proteins at rates proportional to the amount of casein mRNA, but the newly synthesized caseins in cells on plastic are degraded intracellularly, whereas those synthesized by cells on floating gels are secreted into the medium. (v) Cells on all substrata examined lose virtually all mRNA for whey acidic protein despite the fact that this mRNA is abundant in the mammary gland itself; the authors conclude that additional, as-yet-unknown, factors are necessary for synthesis and secretion of whey acidic protein in culture.

  18. Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland

    PubMed Central

    1981-01-01

    A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes