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Sample records for human mammary cell

  1. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    Epithelial Stem Cells PRINCIPAL INVESTIGATOR: Peter D. Eirew CONTRACTING ORGANIZATION: British Columbia Cancer Agency...NUMBER Characterization of Human Mammary Epithelial Stem Cells 5b. GRANT NUMBER W81XWH-06-1-0702 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Abstract The mammary epithelium in normal adult female mice contains undifferentiated stem cells with extensive in vivo regenerative and self-renewal

  2. 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

  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

    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.

  8. 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

  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. 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.

  11. 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.

  12. 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.

  13. 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

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

    DTIC Science & Technology

    2012-04-01

    algorithm for CpG-island detection. BMC Bioinformatics 7: 446. 17. Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol...it does not have a CpG island according to the original criteria (Gardiner-Garden and Frommer 1987). H3K4me3 and H3Ac are present in miR-205...culture of normal human mammary epithelial cells. Cancer Res 69: 7557–7568. Gardiner-GardenM, Frommer M. 1987. CpG islands in vertebrate genomes. J Mol

  15. Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone

    PubMed Central

    Mukhina, Svetlana; Mertani, Hichem C.; Guo, Ke; Lee, Kok-Onn; Gluckman, Peter D.; Lobie, Peter E.

    2004-01-01

    We report here that autocrine production of human growth hormone (hGH) results in a phenotypic conversion of mammary carcinoma cells such that they exhibit the morphological and molecular characteristics of a mesenchymal cell, including expression of fibronectin and vimentin. Autocrine production of hGH resulted in reduced plakoglobin expression and relocalization of E-cadherin to the cytoplasm, leading to dissolution of cell-cell contacts and decreased cell height. These phenotypic changes were accompanied by an increase in cell motility, elevated activity of specific matrix metalloproteinases, and an acquired ability to invade a reconstituted basement membrane. Forced expression of plakoglobin significantly decreased mammary carcinoma cell migration and invasion stimulated by autocrine hGH. In vivo, autocrine hGH stimulated local invasion of mammary carcinoma cells concomitant with a prominent stromal reaction in comparison with well delineated and capsulated growth of mammary carcinoma cells lacking autocrine production of hGH. Thus, autocrine production of hGH by mammary carcinoma cells is sufficient for generation of an invasive phenotype. Therapeutic targeting of autocrine hGH may provide a mechanistic approach to prevent metastatic extension of human mammary carcinoma. PMID:15353581

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

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

    DTIC Science & Technology

    2002-10-01

    Hamann , P. Jenti, B. Imhof, and D. Vestweber. 1993. A death and tissue remodeling during mouse mammary gland involution. De- monoclonal antibody...Cell Growth Differ 10:49-59. Kato J-Y, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ . 1993. Snowden AW, Perkins ND. 1998. Cell cycle regulation of the

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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

  6. 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

  7. BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells

    PubMed Central

    Nemirovsky, Oksana; Chen, Helen; Connell, Marisa; Taylor, Brian; Jiang, Jihong; Pilarski, Linda M.; Fleisch, Markus C.; Niederacher, Dieter; Pujana, Miguel Angel; Eaves, Connie J.; Maxwell, Christopher A.

    2017-01-01

    BRCA1 deficiency may perturb the differentiation hierarchy present in the normal mammary gland and is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome. Oriented cell division is a mechanism known to regulate cell fates and to restrict tumor formation. We now show that the cell division axis is altered following shRNA-mediated BRCA1 depletion in immortalized but non-tumorigenic, or freshly isolated normal human mammary cells with graded consequences in progeny cells that include aneuploidy, perturbation of cell polarity in spheroid cultures, and a selective loss of cells with luminal features. BRCA1 depletion stabilizes HMMR abundance and disrupts cortical asymmetry of NUMA-dynein complexes in dividing cells such that polarity cues provided by cell-matrix adhesions were not able to orient division. We also show that immortalized mammary cells carrying a mutant BRCA1 allele (BRCA1 185delAG/+) reproduce many of these effects but in this model, oriented divisions were maintained through cues provided by CDH1+ cell-cell junctions. These findings reveal a previously unknown effect of BRCA1 suppression on mechanisms that regulate the cell division axis in proliferating, non-transformed human mammary epithelial cells and consequent downstream effects on the mitotic integrity and phenotype control of their progeny. PMID:28427147

  8. BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells.

    PubMed

    He, Zhengcheng; Kannan, Nagarajan; Nemirovsky, Oksana; Chen, Helen; Connell, Marisa; Taylor, Brian; Jiang, Jihong; Pilarski, Linda M; Fleisch, Markus C; Niederacher, Dieter; Pujana, Miguel Angel; Eaves, Connie J; Maxwell, Christopher A

    2017-05-16

    BRCA1 deficiency may perturb the differentiation hierarchy present in the normal mammary gland and is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome. Oriented cell division is a mechanism known to regulate cell fates and to restrict tumor formation. We now show that the cell division axis is altered following shRNA-mediated BRCA1 depletion in immortalized but non-tumorigenic, or freshly isolated normal human mammary cells with graded consequences in progeny cells that include aneuploidy, perturbation of cell polarity in spheroid cultures, and a selective loss of cells with luminal features. BRCA1 depletion stabilizes HMMR abundance and disrupts cortical asymmetry of NUMA-dynein complexes in dividing cells such that polarity cues provided by cell-matrix adhesions were not able to orient division. We also show that immortalized mammary cells carrying a mutant BRCA1 allele (BRCA1 185delAG/+) reproduce many of these effects but in this model, oriented divisions were maintained through cues provided by CDH1+ cell-cell junctions. These findings reveal a previously unknown effect of BRCA1 suppression on mechanisms that regulate the cell division axis in proliferating, non-transformed human mammary epithelial cells and consequent downstream effects on the mitotic integrity and phenotype control of their progeny.

  9. 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

  10. 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.

  11. The CD10 enzyme is a key player to identify and regulate human mammary stem cells.

    PubMed

    Bachelard-Cascales, Elodie; Chapellier, Marion; Delay, Emmanuel; Pochon, Gaetan; Voeltzel, Thibault; Puisieux, Alain; Caron de Fromentel, Claude; Maguer-Satta, Véronique

    2010-06-01

    The major components of the mammary ductal tree are an inner layer of luminal cells, an outer layer of myoepithelial cells, and a basement membrane that separates the ducts from the underlying stroma. Cells in the outer layer express CD10, a zinc-dependent metalloprotease that regulates the growth of the ductal tree during mammary gland development. To define the steps in the human mammary lineage at which CD10 acts, we have developed an in vitro assay for human mammary lineage progression. We show that sorting for CD10 and EpCAM cleanly separates progenitors from differentiated luminal cells and that the CD10-high EpCAM-low population is enriched for early common progenitor and mammosphere-forming cells. We also show that sorting for CD10 enriches sphere-forming cells from other tissue types, suggesting that it may provide a simple tool to identify stem or progenitor populations in tissues for which lineage studies are not currently possible. We demonstrate that the protease activity of CD10 and the adhesion function of beta1-integrin are required to prevent differentiation of mammary progenitors. Taken together, our data suggest that integrin-mediated contact with the basement membrane and cleavage of signaling factors by CD10 are key elements in the niche that maintains the progenitor and stem cell pools in the mammary lineage.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Adiponectin differentially affects gene expression in human mammary epithelial and breast cancer cells.

    PubMed

    Treeck, O; Lattrich, C; Juhasz-Boess, I; Buchholz, S; Pfeiler, G; Ortmann, O

    2008-10-21

    Serum levels of adiponectin are inversely associated with breast cancer risk. In this study, its effect on growth and gene expression of MCF-7 breast cancer cells and MCF-10A human mammary epithelial cells was compared. The antiproliferative effect of adiponectin on MCF-10A cells was more pronounced and was accompanied by elevated transcript levels of caspase 1, ERbeta2, ERbeta5, TR2 and USP2. Our data suggest that upregulation of genes with known growth inhibitory or apoptotic functions in mammary epithelial cells might contribute to the protective action of this adipocytokine.

  17. 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.

  18. Clonal analysis of morphological phenotype in cultured mammary epithelial cells from human milk.

    PubMed

    Stoker, M; Perryman, M; Eeles, R

    1982-05-22

    Three main types of colony forming epithelial cell, termed elongated, cuboidal and open, are found in cultures of human milk. Subculture of identified colonies, and cloning from single cells shows that each cell type can maintain its morphological phenotype, but in addition the cuboidal and open cell types can give rise to the elongated type. The results, which suggest a differentiation pathway starting with open cell types, are discussed in relation to differentiation studies on mammary cancer cells.

  19. 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.

  20. Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

    PubMed Central

    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. PMID:23155468

  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

    satellites ” resulted in reversion of the cells to an epithelial state, re-entry into the cell cycle, and restoration of their ability to generate both...1 AWARD NUMBER: W81XWH-12-1-0107 TITLE: Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer...W81XWH-12-1-0107 Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

  2. 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.

  3. 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

  4. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells.

    PubMed

    Sappino, André-Pascal; Buser, Raphaële; Lesne, Laurence; Gimelli, Stefania; Béna, Frédérique; Belin, Dominique; Mandriota, Stefano J

    2012-03-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics. Copyright © 2012 John Wiley & Sons, Ltd.

  5. 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.

  6. 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.

  7. 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.

  8. 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).

  9. 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).

  10. Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells.

    PubMed

    Zhou, Yang; Ren, Linzhu; Zhu, Jianguo; Yan, Sen; Wang, Haijun; Song, Na; Li, Li; Ouyang, Hongsheng; Pang, Daxin

    2011-08-01

    Human Fibroblast growth factor 1 (FGF1) has been recognized as a valuable protein drug for the treatment of many diseases because of its multiple functions in regulating a variety of biological processes involved in embryonic development, cell growth and differentiation, morphogenesis, tissue repair, and others. The aim of this study was to develop an FGF1 mammary gland-specific expression vector to produce FGF1 on a large scale from transgenic cows to meet the demand for FGF1 in medical use. In this study, we generated an FGF1 mammary gland-specific expression vector and validated its function in human MCF-7 cells. This vector was shown to successfully express functional FGF1, thus potentially enabling the generation of transgenic cows to be used as mammary gland bioreactors.

  11. Exogenous normal mammary epithelial mitochondria suppress glycolytic metabolism and glucose uptake of human breast cancer cells.

    PubMed

    Jiang, Xian-Peng; Elliott, Robert L; Head, Jonathan F

    2015-10-01

    We hypothesized that normal mitochondria inhibited cancer cell proliferation and increased drug sensitivity by the mechanism of suppression of cancer aerobic glycolysis. To demonstrate the mechanism, we used real-time PCR and glycolysis cell-based assay to measure gene expression of glycolytic enzymes and glucose transporters, and extracellular lactate production of human breast cancer cells. We found that isolated fluorescent probe-stained mitochondria of MCF-12A (human mammary epithelia) could enter into human breast cancer cell lines MCF-7, T47D, and MDA-MB-231, confirmed by fluorescent and confocal microscopy. Mitochondria from the untransformed human mammary epithelia increased drug sensitivity of MCF-7 cells to paclitaxel. Real-time PCR showed that exogenous normal mitochondria of MCF-12A suppressed gene expression of glycolytic enzymes, lactate dehydrogenase A, and glucose transporter 1 and 3 of MCF-7 and MDA-MB-231 cells. Glycolysis cell-based assay revealed that normal mitochondria significantly suppressed lactate production in culture media of MCF-7, T47D, and MDA-MB-231 cells. In conclusion, normal mitochondria suppress cancer proliferation and increase drug sensitivity by the mechanism of inhibition of cancer cell glycolysis and glucose uptake.

  12. Mechanistic insights into the cytotoxicity and genotoxicity induced by glycidamide in human mammary cells.

    PubMed

    Bandarra, Susana; Fernandes, Ana S; Magro, Inês; Guerreiro, Patrícia S; Pingarilho, Marta; Churchwell, Mona I; Gil, Octávia Monteiro; Batinic-Haberle, Ines; Gonçalves, Sandrina; Rueff, José; Miranda, Joana P; Marques, M Matilde; Beland, Frederick A; Castro, Matilde; Gaspar, Jorge F; Oliveira, Nuno G

    2013-11-01

    Acrylamide (AA) is a well-known industrial chemical classified as a probable human carcinogen. Benign and malignant tumours at different sites, including the mammary gland, have been reported in rodents exposed to AA. This xenobiotic is also formed in many carbohydrate-rich foods prepared at high temperatures. For this reason, AA is an issue of concern in terms of human cancer risk. The epoxide glycidamide (GA) is thought to be the ultimate genotoxic AA metabolite. Despite extensive experimental and epidemiological data focused on AA-induced breast cancer, there is still lack of information on the deleterious effects induced by GA in mammary cells. The work reported here addresses the characterisation and modulation of cytotoxicity, generation of reactive oxygen species, formation of micronuclei (MN) and quantification of specific GA-DNA adducts in human MCF10A epithelial cells exposed to GA. The results show that GA significantly induces MN, impairs cell proliferation kinetics and decreases cell viability at high concentrations by mechanisms not involving oxidative stress. KU55933, an inhibitor of ataxia telangiectasia mutated kinase, enhanced the cytotoxicity of GA (P < 0.05), supporting a role of this enzyme in regulating the repair of GA-induced DNA lesions. Moreover, even at low GA levels, N7-GA-Gua adducts were generated in a linear dose-response manner in MCF10A cells. These results confirm that human mammary cells are susceptible to GA toxicity and reinforce the need for additional studies to clarify the potential correlation between dietary AA exposure and breast cancer risk in human populations.

  13. 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.

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

    PubMed Central

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

    2011-01-01

    Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMEC), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5), were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrates that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. While normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMEC cells under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. This study discusses some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from the response to acute oxidative stress in normal mammary epithelial cells. PMID:21397008

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

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

    DTIC Science & Technology

    2005-06-01

    ER+ cell lines are sensitive to estrogen, we tested whether 17P3-estradiol can stimulate transcriptional activation of an estrogen- responsive...MSI in sporadic colon cancer is transcriptional silencing of the cDNA. Results show that MMR-proficient cells have hMLHI1 gene by methylation of the...three independent experiments.,0 14) 0 a) The asterisk indicates significantd frence (P • 0-05) from other mecan "- " - - values with student t tests

  2. 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.

  3. 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.

  4. 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).

  5. 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).

  6. 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.

  7. 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.

  8. 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

  9. Carcinogen-induced histone alteration in normal human mammary epithelial cells.

    PubMed

    Bradley, Chastity; van der Meer, Riet; Roodi, Nady; Yan, Heping; Chandrasekharan, Mahesh B; Sun, Zu-Wen; Mernaugh, Ray L; Parl, Fritz F

    2007-10-01

    Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

  10. 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.

  11. 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).

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

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

    PubMed Central

    Sridharan, Deepa M.; Enerio, Shiena; LaBarge, Mark A.; Stampfer, Martha M.; Pluth, Janice M.

    2017-01-01

    Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations and changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population. PMID:28245431

  2. 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

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

    PubMed

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

    2016-06-28

    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.

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

    SciTech Connect

    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. S.; Qian, Weijun

    2009-08-01

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. 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 an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

  7. 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

  8. Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

    PubMed

    Kannan, Nagarajan; Nguyen, Long V; Makarem, Maisam; Dong, Yifei; Shih, Kingsley; Eirew, Peter; Raouf, Afshin; Emerman, Joanne T; Eaves, Connie J

    2014-05-27

    Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

  9. 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

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

    PubMed

    Fedrowitz, Maren; Hass, Ralf; Bertram, Catharina; Löscher, Wolfgang

    2011-01-01

    Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth. The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress marker, is able to exert antiproliferative effects on the growth of mammary gland epithelial cells. For this purpose, primary epithelial cultures of breast tissue from two different inbred rat strains, Fischer 344 (F344) and Lewis, as well as breast tumor cells of human origin were used. Treatment with human salivary α-amylase was performed once daily for 2 days followed by cell counting (trypan blue assay) to determine alterations in cell numbers. Cell senescence after α-amylase treatment was assessed by β-galactosidase assay. Endogenous α-amylase was detected in cells from F344 and Lewis by immunofluorescence. Salivary α-amylase treatment in vitro significantly decreased the proliferation of primary cells from F344 and Lewis rats in a concentration-dependent manner. Noticeably, the sensitivity towards α-amylase was significantly higher in Lewis cells with stronger impact on cell growth after 5 and 50 U/ml compared to F344 cells. An antiproliferative effect of α-amylase was also determined in mammary tumor cells of human origin, but this effect varied depending on the donor, age, and type of the cells. The results presented here indicate for the first time that salivary α-amylase affects cell growth in rat mammary epithelial cells and in breast tumor cells of human origin. Thus, α-amylase may be considered a novel, promising target for balancing cellular growth, which may provide an interesting tool for tumor prophylaxis and treatment.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells.

    PubMed

    Leadon, S A; Sumerel, J; Minton, T A; Tischler, A

    1995-12-01

    In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.

  16. 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

  17. At last: classification of human mammary cells elucidates breast cancer origins.

    PubMed

    Cardiff, Robert D; Borowsky, Alexander D

    2014-02-01

    Current breast cancer classification systems are based on molecular evaluation of tumor receptor status and do not account for distinct morphological phenotypes. In other types of cancer, taxonomy based on normal cell phenotypes has been extremely useful for diagnosis and treatment strategies. In this issue of the JCI, Santagata and colleagues developed a breast cancer classification scheme based on characterization of healthy mammary cells. Reclassification of breast cancer cells and breast cancer tissue microarrays with this system correlated with prognosis better than the standard receptor status designation. This scheme provides a major advance toward our understanding of the origin of the cells in the breast and breast cancers.

  18. Squalene protects against oxidative DNA damage in MCF10A human mammary epithelial cells but not in MCF7 and MDA-MB-231 human breast cancer cells.

    PubMed

    Warleta, Fernando; Campos, María; Allouche, Yosra; Sánchez-Quesada, Cristina; Ruiz-Mora, Jesús; Beltrán, Gabriel; Gaforio, José J

    2010-04-01

    Until now, very little has been known about the antioxidant capacity of squalene and its effect on human breast tumourigenesis. In the present work, we investigated squalene's scavenging properties and its effect on cell proliferation, cell cycle profile, apoptosis, reactive oxygen species (ROS) level and oxidative DNA damage, using human breast cell lines. Our results showed that squalene neither possesses scavenging activity nor significantly alters cell proliferation rates, the cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A), minimally invasive (MDA-MB-231) breast cancer cells, and highly invasive (MCF7) breast cancer cells. However, we found that squalene did exert the following effects on MCF10A epithelial cells in a dose-dependent manner: (a) it decreased intracellular ROS level, (b) it prevented H(2)O(2)-induced oxidative injury, and (c) it protected against oxidative DNA damage. Interestingly, squalene did not exert these effects on MCF7 and MDA-MB-231 cancer cells. Therefore, our data suggest that squalene, found in high amounts in virgin olive oils, could be partially responsible for the lower incidence of breast cancer in populations that consume the Mediterranean diet due to its protective activity against oxidative DNA damage in normal mammary cells. 2010 Elsevier Ltd. All rights reserved.

  19. 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.

  20. 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.

  1. 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.

  2. 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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. 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.

  4. 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.

  5. 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.

  6. Prevention of Human Mammary Carcinogenesis

    DTIC Science & Technology

    1995-06-30

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

  7. A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

    PubMed

    Song, Jiazhe; Xue, Kai; She, Ji; Ding, Fangrong; Li, Song; Shangguan, Rulan; Dai, Yunping; Du, Liying; Li, Ning

    2014-09-10

    The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin(-)CD24(med)CD29(+) cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin(-)CD24(+)CD29(hi)), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin(-)CD24(med)CD29(+) cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin(-)CD24(med)CD29(+) cell population possessed low proportion of stem cells. In summary, heterogeneous Lin(-)CD24(med)CD29(+) cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. ERBB-2 overexpression confers PI 3 ′ kinase-dependent invasion capacity on human mammary epithelial cells

    PubMed Central

    Ignatoski, K M Woods; Maehama, T; Markwart, S M; Dixon, J E; Livant, D L; Ethier, S P

    2000-01-01

    Amplification and overexpression of ERBB-2 in human breast cancer is thought to play a significant role in the progression of the disease; however, its precise role in the aetiology of altered phenotypes associated with human breast cancer is unknown. We have previously shown that exogenous overexpression of ERBB-2 conferred growth factor independence on human mammary epithelial cells. In this study, we show that ERBB-2 overexpression also causes the cells to acquire other characteristics exhibited by human breast cancer cells, such as anchorage-independent growth and invasion capabilities. ERBB-2-induced invasion is dependent on fibronectin and correlates with the down-regulation of cell surface α4 integrin. In addition ERBB-2 co-immunoprecipitates with focal adhesion kinase (FAK) in these cells. We have also shown, by use of exogenously expressed PTEN and by treatment with the PI3′-kinase inhibitor LY294002, that ERBB-2-induced invasion is dependent on the PI3′-kinase pathway; however, PTEN does not dephosphorylate FAK in these cells. © 2000 Cancer Research Campaign PMID:10682681

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Structural Cues from the Tissue Microenvironment Are Essential Determinants of the Human Mammary Epithelial Cell Phenotype

    PubMed Central

    Schmeichel, Karen L.; Weaver, Valerie M.

    2010-01-01

    Historically, the study of normal human breast function and breast disorders has been significantly impaired by limitations inherent to available model systems. Recent improvements in human breast epithelial cell lines and three-dimensional (3-D)3 culture systems have contributed to the development of in vitro model systems that recapitulate differentiated epithelial cell phenotypes with remarkable fidelity. Molecular characterization of these human breast cell models has demonstrated that normal breast epithelial cell behavior is determined in part by the precise interplay that exists between a cell and its surrounding microenvironment. Recent functional studies of integrins in a human model system provide evidence to support the idea that the structural stability afforded by integrin-mediated cell-extracellular matrix interactions is an important determinant of normal cellular behavior, and that alterations in tissue structure can give rise to tumorigenic progression. PMID:10819528

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25352719

  8. 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

  9. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26192111

  11. 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.

  12. (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

  13. (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

  14. Mammary stem cell research in veterinary science: an update.

    PubMed

    Borena, Bizunesh M; Bussche, Leen; Burvenich, Christian; Duchateau, Luc; Van de Walle, Gerlinde R

    2013-06-15

    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.

  15. 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

  16. 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.

  17. Unique growth pattern of human mammary epithelial cells induced by polymeric nanoparticles

    PubMed Central

    Hussien, Rajaa; Rihn, Bertrand H; Eidi, Housam; Ronzani, Carole; Joubert, Olivier; Ferrari, Luc; Vazquez, Oscar; Kaufer, Daniela; Brooks, George A

    2013-01-01

    Due to their unique properties, engineered nanoparticles (NPs) have found broad use in industry, technology, and medicine, including as a vehicle for drug delivery. However, the understanding of NPs’ interaction with different types of mammalian cells lags significantly behind their increasing adoption in drug delivery. In this study, we show unique responses of human epithelial breast cells when exposed to polymeric Eudragit® RS NPs (ENPs) for 1–3 days. Cells displayed dose-dependent increases in metabolic activity and growth, but lower proliferation rates, than control cells, as evidenced in tetrazolium salt (WST-1) and 5-bromo-2′-deoxyuridine (BrdU) assays, respectively. Those effects did not affect cell death or mitochondrial fragmentation. We attribute the increase in metabolic activity and growth of cells culture with ENPs to three factors: (1) high affinity of proteins present in the serum for ENPs, (2) adhesion of ENPs to cells, and (3) activation of proliferation and growth pathways. The proteins and genes responsible for stimulating cell adhesion and growth were identified by mass spectrometry and Microarray analyses. We demonstrate a novel property of ENPs, which act to increase cell metabolic activity and growth and organize epithelial cells in the epithelium as determined by Microarray analysis. PMID:24303146

  18. 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

  19. Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents.

    PubMed

    Cui, Yuxia; Palii, Stela S; Innes, Cynthia L; Paules, Richard S

    2014-01-01

    DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

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

    SciTech Connect

    Nijjar, Tarlochan; Wigington, Don; Garbe, James C.; Waha, Andreas; Stampfer, Martha R.; Yaswen, Paul

    1999-08-01

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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length

    PubMed Central

    2013-01-01

    Background Shortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process. Here we ascertained whether changes in telomere lengths or telomerase activity correlated with age in normal human mammary epithelial cells (HMEC), or with phenotypes of aging in breast. Accordingly, flow cytometry fluorescence in situ hybridization (flowFISH) was used to determine relative telomere lengths (RTL), and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP), in a collection of 41 primary HMEC strains established from women aged 16 to 91 years. Results RTL measurements of HMEC strains that were heterogeneous with respect to lineage composition revealed no significant associations between telomere length with age, maximum observed population doublings, or with lineage composition of the strains. However, within strains, luminal epithelial and cKit-expressing epithelial progenitor cells that were flow cytometry-enriched from individual HMEC strains exhibited significantly shorter telomeres relative to isogenic myoepithelial cells (P < 0.01). In unsorted strains, detectable telomerase activity did not correlate with RTL. Telomerase activity declined with age; the average age of strains that exhibited TRAP activity was 29.7 ± 3.9y, whereas the average age of strains with no detectable TRAP activity was 49.0 ± 4.9y (P < 0.01). Non-detectable TRAP activity also was correlated with phenotypes of aging previously described in HMEC strains; increased proportions of CD227-expressing luminal epithelial cells (P < 0.05) and cKit-expressing progenitor cells (P < 0.05). Conclusions Telomere shortening did not correlate with the chronological ages of HMEC strains, whereas decreased telomerase activity correlated with age and with lineage distribution phenotypes characteristic of aging. PMID:23718190

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Conserved and variable: Understanding mammary stem cells across species.

    PubMed

    Rauner, Gat; Ledet, Melissa M; Van de Walle, Gerlinde R

    2017-08-22

    Postnatal mammary gland development requires the presence of mammary stem and progenitor cells (MaSC), which give rise to functional milk-secreting cells and regenerate the mammary epithelium with each cycle of lactation. These long-lived, tissue-resident MaSC are also targets for malignant transformation and may be cancer cells-of-origin. Consequently, MaSC are extensively researched in relation to their role and function in development, tissue regeneration, lactation, and breast cancer. The basic structure and function of the mammary gland are conserved among all mammalian species, from the most primitive to the most evolved. However, species vary greatly in their lactation strategies and mammary cancer incidence, making MaSC an interesting focus for comparative research. MaSC have been characterized in mice, to a lesser degree in humans, and to an even lesser degree in few additional mammals. They remain uncharacterized in most mammalian species, including "ancient" monotremes, marsupials, wild, and rare species, as well as in common and domestic species such as cats. Identification and comparison of MaSC across a large variety of species, particularly those with extreme lactational adaptations or low mammary cancer incidence, is expected to deepen our understanding of development and malignancy in the mammary gland. Here, we review the current status of MaSC characterization across species, and underline species variations in lactation and mammary cancer through which we may learn about the role of MaSC in these processes. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  14. Boldine Inhibits Mouse Mammary Carcinoma In Vivo and Human MCF-7 Breast Cancer Cells In Vitro.

    PubMed

    Tomšík, Pavel; Mičuda, Stanislav; Muthná, Darina; Čermáková, Eva; Havelek, Radim; Rudolf, Emil; Hroch, Miloš; Kadová, Zuzana; Řezáčová, Martina; Ćmielová, Jana; Živný, Pavel

    2016-11-01

    Boldine is an aporphine alkaloid widely consumed in the folk medicine of some regions. Its anticancer potential has been shown but not yet elucidated. We compared the antitumor effect of orally and parenterally applied boldine in mice bearing solid Ehrlich tumor. We also explored the effects of boldine on breast adenocarcinoma MCF-7 cells in vitro. Repeated i. p. injections of 30, 60, or 90 mg boldine/kg, either alone or combined with doxorubicin, slowed tumor growth in vivo. The latter two doses also prolonged the post-therapeutic survival of the mice. When fed food supplemented with boldine at a dose of 90 mg/kg, the tumor-bearing mice survived significantly longer, but there was no effect on tumor size. Interestingly, continuous p. o. administration did not produce detectable levels of boldine in plasma or tissue samples, in contrast to high but short-lived concentrations after i. p. injections. There was neither antagonism nor synergism between boldine and doxorubicin, except a possible synergism of i. p. boldine 90 mg/kg combined with doxorubicin when compared with doxorubicin alone.Boldine was cytotoxic to MCF-7 cells and reduced their viability and proliferation in vitro. Exposure to boldine decreased bromodeoxyuridine incorporation and histone H3 phosphorylation but did not induce apoptosis. Boldine treatment resulted in p38, ERK, and JNK activation in the mitogen-activated protein kinase pathway in a dose-dependent manner. Since bioavailability in mice seems to be different from that reported in rats, pharmacokinetic studies in humans are needed to evaluate the role of boldine in the beneficial effects of Boldo infusions. Georg Thieme Verlag KG Stuttgart · New York.

  15. 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

  16. Prothrombotic gene expression profile in vascular smooth muscle cells of human saphenous vein, but not internal mammary artery.

    PubMed

    Payeli, S K; Latini, R; Gebhard, C; Patrignani, A; Wagner, U; Lüscher, T F; Tanner, F C

    2008-04-01

    The resistance of internal mammary artery (IMA) toward thrombotic occlusion and accelerated atherosclerosis is not well understood. This study analyzed gene expression profiles of vascular smooth muscle cells (VSMCs) from IMA versus saphenous vein (SV). 54'675 probe sets were examined by Affymetrix microarrays. Thirty-one genes belonged to the coagulation system; 2 were differentially expressed, namely tissue factor (TF) and tissue-type plasminogen activator (tPA). TF was 3.1-fold lower in IMA than SV (P=0.006), whereas tPA was 9.0-fold higher (P<0.001). TF mRNA expression was lower in IMA than SV (P<0.05); tPA was higher (P<0.001). TF protein expression was 4.2+/-0.5-fold lower in IMA than SV (P<0.001); tPA was 2.6+/-0.4-fold higher (P<0.01). In IMA VSMC supernatant, TF protein and activity was lower (P<0.05), TFPI and tPA protein higher (P<0.05 and P<0.005), and clotting time of human plasma prolonged (P<0.05) as compared to SV. Migration to TF/FVIIa (10(-9) mol/L) was 3-fold lower in IMA than SV (P=0.01); PAR-2 protein expression was similar (P=NS), PAR-2 blockade without effect (P=NS). Among the genes of the coagulation system, TF and tPA are differentially expressed in VSMCs from IMA versus SV. This is consistent with protection of IMA from thrombus formation and vascular remodeling.

  17. Geminin overexpression prevents the completion of topoisomerase IIα chromosome decatenation, leading to aneuploidy in human mammary epithelial cells

    PubMed Central

    2011-01-01

    Introduction The nuclear enzyme topoisomerase IIα (TopoIIα) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5′ DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of geminin for improving the therapeutic potential of TopoIIα agents. Methods Human mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoIIα and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase Iε (CKIε) were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7 or CKIε. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoIIα phosphorylation by Cdc7 or CKIε was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively. Results We found that geminin and TopoIIα interact primarily in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa and that geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome

  18. 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.

  19. 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.

  20. 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

  1. 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

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

    DTIC Science & Technology

    2008-08-01

    Cundiff KC, Asch BB: The influence of growth factors on the proliferative potential of normal and primary breast cancer-derived human breast...1755 1946 -2.6 -2.4 -2.1 Entrez [51726]: D 3416 1106 1113 942 -3.1 -3.1 -3.6 Entrez [58515]: S 2835 819 921 815 -3.5 -3.1 -3.5 Entrez [83667]: S 1275

  3. 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.

  4. Expression and Regulation of the Retinoic Acid Receptor Beta Gene in Human Mammary Epithelial Cells

    DTIC Science & Technology

    1996-09-01

    Computer Group, Madison, WI) on the mainframe computer, McClintock at the University of Washington. Results: 68 sequences from 13 breast cancer cells are...promoter sequence (GenBank identifier X56849). 21 Session Name: Barbara Page 1 PileUp of: @total.fil Symbol comparison table- GenR .Dt.. :piltpdnaocmp...Weight: 1.00 Name: 5diDF Len: 433 Check: 598 Weight: 1.00 22 Session Name: Barbara Page 2 Name: 22Di Len: 433 Check: 1929 Weight: 1.00 Name: 16dDF Len

  5. 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

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

    USDA-ARS?s Scientific Manuscript database

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

  7. 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.

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

    PubMed Central

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

    2015-01-01

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

  9. 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.

  10. 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

  11. 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.

  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. Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

    PubMed Central

    Mandel, Katharina; Seidl, Daniel; Rades, Dirk; Lehnert, Hendrik; Gieseler, Frank; Hass, Ralf; Ungefroren, Hendrik

    2013-01-01

    The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor's metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These

  14. 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.

  15. 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.

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

    PubMed Central

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

    2012-01-01

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

  18. Alternative signaling network activation through different insulin receptor family members caused by pro-mitogenic antidiabetic insulin analogues in human mammary epithelial cells.

    PubMed

    ter Braak, Bas; Wink, Steven; Koedoot, Esmee; Pont, Chantal; Siezen, Christine; van der Laan, Jan Willem; van de Water, Bob

    2015-07-19

    Insulin analogues are designed to have improved pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode of action of different insulin analogues. Insulin analogues can bind the insulin receptor and the insulin-like growth factor 1 receptor with different affinities and consequently will activate different downstream signaling pathways. Here we used a panel of MCF7 human breast cancer cell lines that selectively express either one of the isoforms of the INSR or the IGF1R. We applied a transcriptomics approach to assess the differential transcriptional programs activated in these cells by either insulin, IGF1 or X10 treatment. Based on the differentially expressed genes between insulin versus IGF1 and X10 treatment, we retrieved a mitogenic classifier gene set. Validation by RT-qPCR confirmed the robustness of this gene set. The translational potential of these mitogenic classifier genes was examined in primary human mammary cells and in mammary gland tissue of mice in an in vivo model. The predictive power of the classifier genes was evaluated by testing all commercial insulin analogues in the in vitro model and defined X10 and glargine as the most potent mitogenic insulin analogues. We propose that these mitogenic classifier genes can be used to test the mitogenic potential of novel insulin analogues as well as other alternative molecules with an anticipated affinity for the IGF1R.

  19. Prevention of Human Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-07-01

    efficacy of naturally-occurring compounds 13C, EPA, EGCG and GEN was evaluated by determining the down-regulation of the perturbed biomarkers. In the...cells with I3C, EGCG or GEN resulted in differential down-regulation of perturbed biomarkers. This study provides a clinically relevant experimental...naturally-occurring 12 tumor inhibitors 6. Effect of 13C on 184-B5/BP, 184-B5/HER and 13 MDA-MB-231 7. Effect of EGCG on 184-B5/HER 14 8. Effect of GEN on

  20. Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.

    PubMed

    Outzen, H C; Custer, R P

    1975-12-01

    Dysplastic and malignant human breast tissues were grown successfully in the cleared mammary fat pads (CFP) of nude mice. The mammary fat pads were cleared while the mice were in a germfree isolator. Prepared mice were removed fron the germfree enviornment to facilitate transplantation of the human mammary tissue into their CFP and subsequently were maintained in sterile laminar flow racks.

  1. 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

  2. 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

  3. Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts.

    PubMed

    Olsen, Charlotta J; Moreira, José; Lukanidin, Eugene M; Ambartsumian, Noona S

    2010-08-19

    Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s) and cancer cells (MCF7S1) in three-dimensional (3D) growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. In 3D co-culture both HMF3s and MCF7S1 cells demonstrated enhanced invasion into a Matrigel matrix. This was correlated with enhanced expression of the metastasis promoting S100A4 protein in fibroblasts, stimulation of the matrix metalloproteinase (MMP)-2 activity, and enhanced secretion of a range of different cytokines. Orthotopic injection of oestrogen-dependent MCF7S1 cancer cells together with fibroblasts showed stimulation of tumour growth in mice without an external oestrogen supply. The resulting tumours were characterized by increased development of extracellular matrix, as well as an increase of murine S100A4 concentration and activity of MMP-2 in the tumour interstitial fluid. Stimulation of the invasive phenotype of tumour cells in 3D co-cultures with fibroblasts could be correlated with increased production of S100A4 and MMP-2. We propose that enhanced development of mouse host-derived tumour stroma in a MCF7S1 co-injection xenograft model leads to oestrogen independency and is triggered by the initial presence of human fibroblasts.

  4. trans-11 18:1 vaccenic acid (TVA) has a direct anti-carcinogenic effect on MCF-7 human mammary adenocarcinoma cells.

    PubMed

    Lim, Ji-Na; Oh, Jin-Ju; Wang, Tao; Lee, Jae-Sung; Kim, Sang-Hun; Kim, Yoon-Jin; Lee, Hong-Gu

    2014-02-10

    Trans vaccenic acid (TVA; trans-11 18:1) is a positional and geometric isomer of oleic acid and it is the predominant trans isomer found in ruminant fats. TVA can be converted into cis-9, trans-11 conjugated linoleic acid (c9, t11-CLA), a CLA isomer that has many beneficial effects, by stearoyl CoA desaturase 1 (SCD1) in the mammary gland. The health benefits associated with CLA are well documented, but it is unclear whether trans fatty acids (TFAs) from ruminant products have healthy effects. Therefore, the effects of TVA on the proliferation of MCF-7 human breast adenocarcinoma cells and MCF-10A human breast epithelial cells were investigated in the present study. Results showed that TVA inhibited the proliferation of MCF-7 cells but not MCF-10A cells by down-regulating the expression of Bcl-2 as well as procaspase-9. In addition, the suppressive effect of TVA was confirmed in SCD1-depleted MCF-7 cells. Our results suggested that TVA exerts a direct anti-carcinogenic effect on MCF-7 cells. These findings provided a better understanding of the research on the anti-carcinogenic effects of TVA and this may facilitate the manufacture of TVA/c9, t11-CLA fortified ruminant products.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Mammary fibroadenoma with pleomorphic stromal cells.

    PubMed

    Abid, Najla; Kallel, Rim; Ellouze, Sameh; Mellouli, Manel; Gouiaa, Naourez; Mnif, Héla; Boudawara, Tahia

    2015-01-01

    The presence of enlarged and pleomorphic nuclei is usually regarded as a feature of malignancy, but it may on occasion be seen in benign lesions such as mammary fibroadenomas. We present such a case of fibroadenoma occurring in a 37-year-old woman presenting with a self-palpable right breast mass. Histological examination of the tumor revealed the presence of multi and mononucleated giant cells with pleomorphic nuclei. The recognition of the benign nature of these cells is necessary for differential diagnosis from malignant lesions of the breast. fibroadenoma - pleomorphic stromal cells - atypia - breast.

  11. 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.

  12. Migrastatin Analogues Inhibit Canine Mammary Cancer Cell Migration and Invasion

    PubMed Central

    Majchrzak, Kinga; Lo Re, Daniele; Gajewska, Małgorzata; Bulkowska, Małgorzata; Homa, Agata; Pawłowski, Karol; Motyl, Tomasz; Murphy, Paul V.; Król, Magdalena

    2013-01-01

    Background Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. Results Our results showed that two of six fully synthetic analogues of migrastatin: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. Conclusion Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further in

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

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

    PubMed Central

    Visvader, Jane E.; Stingl, John

    2014-01-01

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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. EGFR signaling pathways are wired differently in normal 184A1L5 human mammary epithelial and MDA-MB-231 breast cancer cells.

    PubMed

    Speth, Zachary; Islam, Tanzila; Banerjee, Kasturi; Resat, Haluk

    2017-03-29

    Because of differences in the downstream signaling patterns of its pathways, the role of the human epidermal growth factor family of receptors (HER) in promoting cell growth and survival is cell line and context dependent. Using two model cell lines, we have studied how the regulatory interaction network among the key proteins of HER signaling pathways may be rewired upon normal to cancerous transformation. We in particular investigated how the transcription factor STAT3 and several key kinases' involvement in cancer-related signaling processes differ between normal 184A1L5 human mammary epithelial (HME) and MDA-MB-231 breast cancer epithelial cells. Comparison of the responses in these cells showed that normal-to-cancerous cellular transformation causes a major re-wiring of the growth factor initiated signaling. In particular, we found that: i) regulatory interactions between Erk, p38, JNK and STAT3 are triangulated and tightly coupled in 184A1L5 HME cells, and ii) STAT3 is only weakly associated with the Erk-p38-JNK pathway in MDA-MB-231 cells. Utilizing the concept of pathway substitution, we predicted how the observed differences in the regulatory interactions may affect the proliferation/survival and motility responses of the 184A1L5 and MDA-MB-231 cells when exposed to various inhibitors. We then validated our predictions experimentally to complete the experiment-computation-experiment iteration loop. Validated differences in the regulatory interactions of the 184A1L5 and MDA-MB-231 cells indicated that instead of inhibiting STAT3, which has severe toxic side effects, simultaneous inhibition of JNK together with Erk or p38 could be a more effective strategy to impose cell death selectively to MDA-MB-231 cancer cells while considerably lowering the side effects to normal epithelial cells. Presented analysis establishes a framework with examples that would enable cell signaling researchers to identify the signaling network structures which can be used to

  9. Cell proliferation and apoptosis in rat mammary glands following combinational exposure to bisphenol A and genistein.

    PubMed

    Wang, Jun; Jenkins, Sarah; Lamartiniere, Coral A

    2014-05-29

    Humans are exposed to an array of both harmful and beneficial hormonally active compounds in the environment and through diet. Two such chemicals are Bisphenol A (BPA), a plasticizer, and genistein, a component of soy. Prepubertal exposure to BPA increased mammary carcinogenesis, while genistein suppressed cancer in a chemically-induced model of rodent mammary cancer. The purpose of this research was to determine the effects of combinational exposure to genistein and BPA on cell proliferation, apoptosis, and associated proteins as markers of cancer in mammary glands of rats exposed prepubertally to these environmental chemicals. Prepubertal rats (postpartum days (PND) 2-20) were exposed through lactation via nursing dams treated orally with sesame oil (SO), BPA, genistein, or a combination of BPA and genistein (BPA + Gen). Cell proliferation, apoptosis and protein expressions were investigated for mechanistic studies in mammary glands of rats exposed to these environmental chemicals. Prepubertal exposure to genistein increased cell proliferation in mammary glands of PND21 rats, while BPA increased cell proliferation in adult (PND50) rats. Prepubertal combinational exposure to BPA + Gen increased cell proliferation and reduced apoptosis in PND21 rats, but reduced cell proliferation and increased apoptosis in PND50 rats. The altered mechanisms behind these cellular responses appear to be centered on differential protein expression of caspases, PARP, Bad, p21, Akts, PTEN, ER-β and SRCs 1-3, in the rat mammary gland. Prepubertal BPA exposure resulted in increased cell proliferation in mammary glands of PND50 rats, a process associated with increased risk of cancer development in a chemically-induced mammary cancer. On the other hand, genistein stimulated cell proliferation at PND21, a process that correlates with mammary gland maturation and chemoprevention. In contrast to single chemical exposure, combinational exposure to BPA + Gen performed most similarly to

  10. Cell proliferation and apoptosis in rat mammary glands following combinational exposure to bisphenol A and genistein

    PubMed Central

    2014-01-01

    Background Humans are exposed to an array of both harmful and beneficial hormonally active compounds in the environment and through diet. Two such chemicals are Bisphenol A (BPA), a plasticizer, and genistein, a component of soy. Prepubertal exposure to BPA increased mammary carcinogenesis, while genistein suppressed cancer in a chemically-induced model of rodent mammary cancer. The purpose of this research was to determine the effects of combinational exposure to genistein and BPA on cell proliferation, apoptosis, and associated proteins as markers of cancer in mammary glands of rats exposed prepubertally to these environmental chemicals. Methods Prepubertal rats (postpartum days (PND) 2–20) were exposed through lactation via nursing dams treated orally with sesame oil (SO), BPA, genistein, or a combination of BPA and genistein (BPA + Gen). Cell proliferation, apoptosis and protein expressions were investigated for mechanistic studies in mammary glands of rats exposed to these environmental chemicals. Results Prepubertal exposure to genistein increased cell proliferation in mammary glands of PND21 rats, while BPA increased cell proliferation in adult (PND50) rats. Prepubertal combinational exposure to BPA + Gen increased cell proliferation and reduced apoptosis in PND21 rats, but reduced cell proliferation and increased apoptosis in PND50 rats. The altered mechanisms behind these cellular responses appear to be centered on differential protein expression of caspases, PARP, Bad, p21, Akts, PTEN, ER-β and SRCs 1–3, in the rat mammary gland. Conclusion Prepubertal BPA exposure resulted in increased cell proliferation in mammary glands of PND50 rats, a process associated with increased risk of cancer development in a chemically-induced mammary cancer. On the other hand, genistein stimulated cell proliferation at PND21, a process that correlates with mammary gland maturation and chemoprevention. In contrast to single chemical exposure, combinational exposure to

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Developmental biology: cell fate in the mammary gland

    USDA-ARS?s Scientific Manuscript database

    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. ...

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Differential expression of CD66a (BGP), a cell adhesion molecule of the carcinoembryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland.

    PubMed

    Riethdorf, L; Lisboa, B W; Henkel, U; Naumann, M; Wagener, C; Löning, T

    1997-07-01

    CD66a, also known as biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and the human homologue of the rat cell-CAM. There is evidence that aberrant expression or loss of CD66a in tumor tissue is of biological significance. No data about its expression in breast carcinoma cells and only sparse information about the expression of CD66a in normal breast are available thus far. In this study we used monoclonal antibodies to analyze the expression of CD66a and CEA in normal tissue, benign lesions, and in noninvasive and invasive carcinomas of the mammary gland. In normal tissue and benign lesions, CD66a was consistently expressed at the apical sites of epithelial cells and in myoepithelia, whereas CEA was absent or was restricted only to some apical membranes within the ductal tree. The specific staining of myoepithelia was most evident in pseudoinfiltrative radial scars and sclerosing adenosis. However, the apical expression of CD66a disappeared with the development of the malignant phenotype in noninvasive and invasive carcinomas, and changed gradually from low- to high-grade noninvasive carcinomas into a predominant uniform membrane staining all around the atypical cells. CEA expression was irregular in intensity and distribution. The native apical CD66a staining was partially preserved in some highly differentiated invasive carcinomas with a better prognosis, such as tubular and papillary carcinomas. These findings indicate that loss of CD66a expression rather than a change in staining patterns coincides with the development of the malignant phenotype.

  5. 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.

  6. 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.

  7. 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.

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

    PubMed Central

    Haricharan, S; Li, Y

    2013-01-01

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

  9. 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.

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

    PubMed

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

    2016-12-01

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

  11. 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.

  12. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  14. 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.

  15. 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.

  16. 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

  17. Genome-Wide Small RNA Sequencing and Gene Expression Analysis Reveals a microRNA Profile of Cancer Susceptibility in ATM-Deficient Human Mammary Epithelial Cells

    PubMed Central

    Hesse, Jill E.; Liu, Liwen; Innes, Cynthia L.; Cui, Yuxia; Palii, Stela S.; Paules, Richard S.

    2013-01-01

    Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a syndrome characterized by neurological, motor and immunological defects, and a predisposition to cancer. MicroRNAs (miRNAs) are useful tools for cancer profiling and prediction of therapeutic responses to clinical regimens. We investigated the consequences of ATM deficiency on miRNA expression and associated gene expression in normal human mammary epithelial cells (HME-CCs). We identified 81 significantly differentially expressed miRNAs in ATM-deficient HME-CCs using small RNA sequencing. Many of these have been implicated in tumorigenesis and proliferation and include down-regulated tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as over-expressed pro-oncogenic miRNAs, such as hsa-miR-93 and hsa-miR-221. MicroRNA changes were integrated with genome wide gene expression profiles to investigate possible miRNA targets. Predicted mRNA targets of the miRNAs significantly regulated after ATM depletion included many genes associated with cancer formation and progression, such as SOCS1 and the proto-oncogene MAF. While a number of miRNAs have been reported as altered in cancerous cells, there is little understanding as to how these small RNAs might be driving cancer formation or how they might be used as biomarkers for cancer susceptibility. This study provides preliminary data for defining miRNA profiles that may be used as prognostic or predictive biomarkers for breast cancer. Our integrated analysis of miRNA and mRNA expression allows us to gain a better understanding of the signaling involved in breast cancer predisposition and suggests a mechanism for the breast cancer-prone phenotype seen in ATM-deficient patients. PMID:23741392

  18. Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies

    PubMed Central

    2010-01-01

    Introduction Most human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated β-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth. Methods Pre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or γ-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated β-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses. Results Cultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated β-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent

  19. TCDD exposure disrupts mammary epithelial cell differentiation and function

    PubMed Central

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

    2011-01-01

    Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced β-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less alveolar-like structure formation. SCp2 cells express transcriptionally active AhR, and exposure to TCDD induces expression of the AhR target gene CYP1B1. Exposure to TCDD during pregnancy reduced expression of the cell adhesion molecule E-cadherin in the mammary gland and decreased phosphorylation of STAT5, a known regulator of β-casein gene expression. These data provide morphological and molecular evidence that TCDD-mediated AhR activation disrupts structural and functional differentiation of the mammary gland, and present an in vitro model for studying the effects of TCDD on mammary epithelial cell function. PMID:19490989

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Precursors of hexoneogenesis within the human mammary gland

    USDA-ARS?s Scientific Manuscript database

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

  7. NAD(P)H:quinone oxidoreductase 1 Arg139Trp and Pro187Ser polymorphisms imbalance estrogen metabolism towards DNA adduct formation in human mammary epithelial cells.

    PubMed

    Singh, Seema; Zahid, Muhammad; Saeed, Muhammad; Gaikwad, Nilesh W; Meza, Jane L; Cavalieri, Ercole L; Rogan, Eleanor G; Chakravarti, Dhrubajyoti

    2009-10-01

    Estrogens (estrone, E(1); estradiol, E(2)) are oxidized in the breast first to catechols and then to form two ortho-quinones (E(1/2)-3,4-Q) that react with DNA to form depurinating adducts, which lead to mutations associated with breast cancer. NAD(P)H:quinone oxidoreductase 1 (NQO1) reduces these quinones back to catechols, and thus may protect against this mechanism. We examined whether the inheritance of two polymorphic variants of NQO1 (Pro187Ser or Arg139Trp) would result in poor reduction of E(1/2)-3,4-Q in normal human mammary epithelial cells (MCF-10F) and increased depurinating adduct formation. An isogenic set of stably transfected normal human breast epithelial cells (MCF-10F) that express a truncated (135Stop), the wild-type, the 139Trp variant or the 187Ser variant of human NQO1 cDNA was constructed. MCF-10F cells showed a low endogenous NQO1 activity. NQO1 expression was examined by RT-PCR and Western blotting, and catalytic activity of reducing E(2)-3,4-Q to 4-hydroxyE(1/2) and associated changes in the levels of quinone conjugates (4-methoxyE(1/2), 4-OHE(1/2)-2-glutathione, 4-OHE(1/2)-2-Cys and 4-OHE(1/2)-2-N-acetylcysteine) and depurinating DNA adducts (4-OHE(1/2)-1-N3Ade and 4-OHE(1/2)-1-N7Gua) were examined by HPLC with electrochemical detection, as well as by ultra-performance liquid chromatography with tandem mass spectrometry. The polymorphic variants transcribed comparably to the wild-type NQO1, but produced approximately 2-fold lower levels of the protein, suggesting that the variant proteins may become degraded. E(1/2)-3,4-Q toxicity to MCF-10F cells (IC50=24.74 microM) was increased (IC50=3.7 microM) by Ro41-0960 (3 microM), a catechol-O-methyltransferase inhibitor. Cells expressing polymorphic NQO1 treated with E(2)-3,4-Q with or without added Ro41-0960, showed lower ability to reduce the quinone ( approximately 50% lower levels of the free catechols and approximately 3-fold lower levels of methylated catechols) compared to the wild

  8. 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

  9. 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

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

    PubMed

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

    2011-01-01

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

  11. 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

  12. 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

  13. 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

    decreases in Sox10 mRNA expres- sion levels (Figure 1C). Similarly, by using a Sox10-H2BVenus bacterial artificial chromosome (BAC) transgenic mouse...frequencies ([70 %) and widespread chromosomal instability [10]. These data could suggest that the normal LumProg cell is somehow extremely dependent on

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

    DTIC Science & Technology

    2007-06-01

    Argani P, Marks J, Richardson A, Cooper A, Strausberg R, Riggins GJ, Schnitt S, Gabrielson E, Gelman R, Polyak K: Molecular markers in ductal carcinoma in... Polyak K, Wong JS, Lester SC, Kaelin CM: Ductal Carci- noma in Situ of the Breast. New England Journal of Medicine 2004, 350:1430-1441. 71. Baxter...senescence. Cancer Cell 2006; 10:459-72. 9. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV , Kolettas E, Niforou K

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

    DTIC Science & Technology

    2008-06-01

    Cancer Cell 2006; 10:459-72. 9. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV , Kolettas E, Niforou K, Zoumpourlis...Keshaviah A, Bae YK, Argani P, Marks J, Richardson A, Cooper A, Strausberg R, Riggins GJ, Schnitt S, Gabrielson E, Gelman R, Polyak K: Molecular markers in...3326. 70. Burstein HJ, Polyak K, Wong JS, Lester SC, Kaelin CM: Ductal Carci- noma in Situ of the Breast. New England Journal of Medicine 2004, 350

  17. Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCA1 Genes

    DTIC Science & Technology

    1999-01-01

    development of breast cancers. To study the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway, we have...the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway. The consequences of transduction of these...proposed three approaches for constructing p53-deficient cells; i.e., by mutating the p53 gene directly, by abrogating the protein’s normal cellular

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    PubMed

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-08

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1. Copyright © 2016 Elsevier Inc. All rights reserved.

  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. 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.

  5. Mice expressing myrAKT1 in the mammary gland develop carcinogen-induced ER-positive mammary tumors that mimic human breast cancer.

    PubMed

    Blanco-Aparicio, Carmen; Pérez-Gallego, Lucía; Pequeño, Belén; Leal, Juan F M; Renner, Oliver; Carnero, Amancio

    2007-03-01

    AKT1/PKB is a serine/threonine protein kinase that regulates biological processes such as proliferation, apoptosis and growth in a variety of cell types. To assess the oncogenic capability of an activated form of AKT in vivo we have generated several transgenic mouse lines that overexpress in the mammary epithelium the murine Akt1 gene modified with a myristoylation signal, which renders active this protein by localizing it to the plasma membrane. We demonstrate that expression of myristoylated AKT in the mammary glands increases the susceptibility of these mice to the induction of mammary tumors of epithelial origin by the carcinogen 9,10-dimethyl-1,2 benzanthracene (DMBA). We have found that while carcinogen-treated wild-type mice show mostly mammary tumors of sarcomatous origin, AKT transgenic mice treated with DMBA developed mainly adenocarcinoma or adenosquamous tumors, all of them displaying activated AKT. We analyzed other possible molecular alterations cooperating with AKT and found that neither Ras nor beta-catenin/Wnt pathways seemed altered nor p53 mutated. We have found that 100% of mammary DMBA-induced tumors and benign lesions in myrAKT mice are estrogen receptor (ERalpha)-positive and are more frequent than in wild-type littermates. These data show that AKT activation cooperates with deregulation of the estrogen receptor in the DMBA-induced mammary tumorigenesis model and recapitulate two characteristics of some human breast tumors. Thus, our model might provide a preclinical relevant model system to study the role of AKT and ERalpha in breast tumorigenesis and the response of mammary gland tumors to chemotherapeutics.

  6. 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.

  7. 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

  8. Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis.

    PubMed

    Ferreira, Enio; Gobbi, Helenice; Saraiva, Bruna S; Cassali, Geovanni D

    2010-02-23

    It has been suggested that columnar cell lesions indicate an alteration of the human mammary gland involved in the development of breast cancer. They have not previously been described in canine mammary gland. The aim of this paper is describe the morphologic spectrum of columnar cell lesions in canine mammary gland specimens and their association with other breast lesions. A total of 126 lesions were subjected to a comprehensive morphological review based upon the human breast classification system for columnar cell lesions. The presence of preinvasive (epithelial hyperplasia and in situ carcinoma) and invasive lesions was determined and immunophenotypic analysis (estrogen receptor (ER), progesterone receptor (PgR), high molecular weight cytokeratin (34betaE-12), E-cadherin, Ki-67, HER-2 and P53) was perfomed. Columnar cell lesions were identified in 67 (53.1%) of the 126 canine mammary glands with intraepithelial alterations. They were observed in the terminal duct lobular units and characterized at dilated acini may be lined by several layers of columnar epithelial cells with elongated nuclei. Of the columnar cell lesions identified, 41 (61.2%) were without and 26 (38.8%) with atypia. Association with ductal hyperplasia was observed in 45/67 (67.1%). Sixty (89.5%) of the columnar cell lesions coexisted with neoplastic lesions (20 in situ carcinomas, 19 invasive carcinomas and 21 benign tumors). The columnar cells were ER, PgR and E-cadherin positive but negative for cytokeratin 34betaE-12, HER-2 and P53. The proliferation rate as measured by Ki-67 appeared higher in the lesions analyzed than in normal TDLUs. Columnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions.

  9. 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.

  10. The caM kinase, Pnck, is spatially and temporally regulated during murine mammary gland development and may identify an epithelial cell subtype involved in breast cancer.

    PubMed

    Gardner, H P; Ha, S I; Reynolds, C; Chodosh, L A

    2000-10-01

    While screening for protein kinases expressed in the murine mammary gland, we identified previously a Ca2+/calmodulin-dependent kinase, Pnck, that is most closely related to CaMKI. In this report, we show that Pnck is temporally regulated during murine mammary development with highest levels of expression observed late in pregnancy, concomitant with the decreased cellular proliferation and terminal differentiation of the mammary epithelium. Consistent with this finding, Pnck is up-regulated in confluent mammary epithelial cells and is down-regulated as serum-starved cells are stimulated to reenter the cell cycle. In the mammary gland, Pnck is expressed in an epithelial-specific and markedly heterogeneous manner, suggesting that the expression of this kinase may be restricted to a particular mammary epithelial cell type. Potentially related to its heterogeneous in vivo expression pattern, Pnck expression is oncogene-associated in murine epithelial cell lines derived from mammary tumors arising in different transgenic mouse models of breast cancer; cell lines derived from mammary tumors initiated by c-myc or int-2/Fgf3 express Pnck, whereas cell lines initiated by neu or H-ras do not. In an analogous manner, expression of the human homologue of Pnck is restricted to a subset of human breast cancer cell lines. Moreover, PNCK was found to be highly overexpressed in a subset of human primary human breast cancers compared with benign mammary tissue. Together, our data suggest that Pnck may play a role in mammary development, and that expression of this kinase may be restricted to a mammary epithelial cell type that is transformed in a subset of human breast cancers.

  11. 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

  12. 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

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

    2014-11-01

    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 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/10(8) 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 NQO1 protein, or NQO1

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. 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.

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

    USDA-ARS?s Scientific Manuscript database

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

  5. Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A

    PubMed Central

    Murray, Tessa J; Yang, Xinhai; Sherr, David H

    2006-01-01

    Introduction This study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER-, AhRhigh line, Hs578T, were studied. Methods AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, α-naphthoflavone (α-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by 3H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting. Results Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of α-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC50 = 11 μM). Galangin inhibited transition of cells from the G0/G1 to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed. Conclusion Galangin is a strong

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. [Generation of transgenic mice expressing human lysozyme in mammary gland].

    PubMed

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang

    2005-10-01

    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

  16. 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

  17. 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.

  18. Synergistic activity of recombinant human endostatin in combination with adriamycin: analysis of in vitro activity on endothelial cells and in vivo tumor progression in an orthotopic murine mammary carcinoma model.

    PubMed

    Plum, Stacy M; Hanson, Arthur D; Volker, Kirk M; Vu, Hong A; Sim, B Kim Lee; Fogler, William E; Fortier, Anne H

    2003-10-01

    Current combination treatment strategies in malignancy are designed to evaluate the use of cytotoxic drugs and antiangiogenic agents. Endostatin, a fragment of collagen XVIII, specifically inhibits proliferation, migration, and differentiation of endothelial cells in vitro as well as angiogenesis and tumor progression in in vivo models. In this study, we determine the antitumor effect of rhEndostatin administered alone or in combination with Adriamycin against established orthotopic murine mammary carcinoma. Mice bearing orthotopically established DA-3 mammary adenocarcinoma tumors received varying doses of rhEndostatin alone and in combination with Adriamycin to assess tumor growth inhibition. Additional studies of this in vivo combination included a determination of Adriamycin-induced cardiotoxicity and in vitro effects on human umbilical vein endothelial cell proliferation and cord formation. For single-agent activity, optimal tumor growth inhibition was observed after s.c. administration of 50 mg/kg/day rhEndostatin or 5 mg/kg Adriamycin injected i.v. every 4 days. Combination of Adriamycin with optimal or suboptimal doses of rhEndostatin resulted in synergistic inhibition of DA-3 tumor growth. Importantly, unlike other antiangiogenic agents, rhEndostatin did not exacerbate the cardiotoxicity of Adriamycin. The synergistic interaction between rhEndostatin and Adriamycin was also observed in vitro for inhibition of human umbilical vein endothelial cell proliferation and inhibition of cord formation. These data suggest that the synergy observed with rhEndostatin in combination with Adriamycin is exerted at the level of the endothelial cell and can result in enhanced tumor growth inhibition. The potential benefit of Adriamycin used in combination with rhEndostatin is being considered for clinical evaluation.

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

    USDA-ARS?s Scientific Manuscript database

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

  20. 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.

  1. Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

    PubMed Central

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

    2014-01-01

    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. PMID:24572294

  2. 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.

  3. 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

  4. 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.

  5. Influence of fatty acid diets on gene expression in rat mammary epithelial cells.

    PubMed

    Medvedovic, M; Gear, R; Freudenberg, J M; Schneider, J; Bornschein, R; Yan, M; Mistry, M J; Hendrix, H; Karyala, S; Halbleib, D; Heffelfinger, S; Clegg, D J; Anderson, M W

    2009-06-10

    This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

  6. 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

  7. 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.

  8. 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

  9. Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy.

    PubMed

    Dumont, Nancy; Crawford, Yongping G; Sigaroudinia, Mahvash; Nagrani, Shefali S; Wilson, Matthew B; Buehring, Gertrude C; Turashvili, Gulisa; Aparicio, Samuel; Gauthier, Mona L; Fordyce, Colleen A; McDermott, Kimberly M; Tlsty, Thea D

    2009-01-01

    We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic

  10. Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

    PubMed Central

    2009-01-01

    Introduction We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. Methods HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). Results vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. Conclusions We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary

  11. 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.

  12. 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.

  13. 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.

  14. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis.

    PubMed

    Ewald, Andrew J; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S; Werb, Zena

    2008-04-01

    Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly, suggesting common mechanisms of epithelial growth.

  15. 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.

  16. 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.

  17. 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

  18. Mammary epithelial cell transformation: insights from cell culture and mouse models.

    PubMed

    Dimri, Goberdhan; Band, Hamid; Band, Vimla

    2005-01-01

    Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely -- a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16INK4a, p21, p27 and p57), p14ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere

  19. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-12-01

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

  2. 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.

  3. 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.

  4. Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium

    PubMed Central

    2014-01-01

    Introduction Although estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER–, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes. Methods We used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER– mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells. Results We describe a cellular hierarchy in the normal human mammary gland in which ER–/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis. Conclusions This study presents direct evidence that ER+ cells are generated by ER–/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and

  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. 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

  7. 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

  8. 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

  9. Effects of Age and Parity on Mammary Gland Lesions and Progenitor Cells in the FVB/N-RC Mice

    PubMed Central

    Raafat, Ahmed; Strizzi, Luigi; Lashin, Karim; Ginsburg, Erika; McCurdy, David; Salomon, David; Smith, Gilbert H.; Medina, Daniel; Callahan, Robert

    2012-01-01

    The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3–4 pregnancies) females were euthanized at 16–22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer. PMID:22952723

  10. 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.

  11. 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.

  12. Caveolin-1 expression is elevated in claudin-low mammary tumor cells.

    PubMed

    Thompson, Devan E; Siwicky, Megan D; Moorehead, Roger A

    2012-02-22

    Caveolin-1 is a scaffolding protein found in plasma membrane invaginations known as caveolae. Caveolin-1 can regulate a number of intracellular processes such as signal transduction, cholesterol metabolism and vesicular transport. With respect to breast cancer caveolin-1 has been observed in both tumor cells and stromal cells surrounding tumors however most of the recent research has focused on how the loss of caveolin-1 in the stromal cells surrounding the tumor alters the tumor microenvironment. Caveolin-1 expression was evaluated in (1) mammary tumors induced by the transgenic overexpression of the type I insulin-like growth factor receptor (IGF-IR), (2) mammary tumors that became independent of IGF-IR signalling and acquired a claudin-low genotype, (3) two murine mammary epithelial tumor cell lines and (4) two murine mammary claudin-low tumor cell lines. We found that mammary tumors induced by IGF-IR overexpression expressed low levels of caveolin-1 while mammary tumors that became independent of IGF-IR signalling expressed considerably higher levels of caveolin-1. Interestingly, pockets of caveolin-1 positive cells could be observed in some of the IGF-IR-induced mammary tumors and these caveolin-1 positive cells were associated with tumor cells that expressed basal cytokeratins (cytokeratins 5 and 14). This caveolin-1 expression pattern was maintained in the murine mammary tumor cell lines in that the epithelial mammary tumor cell lines expressed little or no caveolin-1 while the claudin-low cell lines expressed caveolin-1. Our model indicates that mammary tumor cells with epithelial characteristics lack caveolin-1 while mesenchymal tumor cells express caveolin-1 suggesting that caveolin-1 may serve as a marker of mammary tumor cells with mesenchymal characteristics such as claudin-low breast tumors.

  13. 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.

  14. 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.

  15. Invasiveness and Ploidy of Human Mammary Carcinomas in Short-Term Culture

    NASA Astrophysics Data System (ADS)

    Smith, Helene S.; Liotta, Lance A.; Hancock, Miriam C.; Wolman, Sandra R.; Hackett, Adeline J.

    1985-03-01

    Invasiveness and ploidy were examined in cultures of human epithelial cells derived from nonmalignant breast tissue, primary breast carcinomas, and breast cancer effusion metastases. Successful short-term culture was achieved from approximately 70% of the primary breast cancers. These primary cancers were essentially diploid by flow cytometry and karyotype in contrast to the effusion metastases, which were mostly aneuploid. The diploid tumor cells retained their malignant phenotype in culture as demonstrated by invasion into a denuded human amnion basement membrane. In contrast, epithelial cells cultured from nonmalignant mammary tissue did not invade the amnion. We suggest that the diploid carcinoma cultures may be useful for investigating the essential differences between normal and malignant cells and may complement information derived from studies of tumor cell lines with grossly aberrant karyotypes.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Progenitor Cell Fate Decisions in Mammary Tumorigenesis

    DTIC Science & Technology

    2012-03-01

    Double-strand break repair is mediated by two major repair pathways, homologous recombination (HR) or nonhomologous end joining ( NHEJ ). In mammalian...cells more than 90% of double-strand breaks are repaired by NHEJ . Impairment of these pathways is associated with cell cycle arrest, cell death

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Persistence of γ-H2AX and 53BP1 foci in proliferating and non-proliferating human mammary epithelial cells after exposure to γ-rays or iron ions.

    PubMed

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

    2011-07-01

    To investigate γ-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 ionising 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 γ-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 γ-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 γ-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. The disappearance of radiation-induced γ-H2AX and 53BP1 foci in HMEC has 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 γ-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 remodelling.

  7. 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.

  8. Mammary Tumor-Associated RNAs Impact Tumor Cell Proliferation, Invasion, and Migration.

    PubMed

    Diermeier, Sarah D; Chang, Kung-Chi; Freier, Susan M; Song, Junyan; El Demerdash, Osama; Krasnitz, Alexander; Rigo, Frank; Bennett, C Frank; Spector, David L

    2016-09-27

    Long non-coding RNAs (lncRNAs) represent the largest and most diverse class of non-coding RNAs, comprising almost 16,000 currently annotated transcripts in human and 10,000 in mouse. Here, we investigated the role of lncRNAs in mammary tumors by performing RNA-seq on tumor sections and organoids derived from MMTV-PyMT and MMTV-Neu-NDL mice. We identified several hundred lncRNAs that were overexpressed compared to normal mammary epithelium. Among these potentially oncogenic lncRNAs we prioritized a subset as Mammary Tumor Associated RNAs (MaTARs) and determined their human counterparts, hMaTARs. To functionally validate the role of MaTARs, we performed antisense knockdown and observed reduced cell proliferation, invasion, and/or organoid branching in a cancer-specific context. Assessing the expression of hMaTARs in human breast tumors revealed that 19 hMaTARs are significantly upregulated and many of these correlate with breast cancer subtype and/or hormone receptor status, indicating potential clinical relevance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Expression and Function of the Protein Tyrosine Phosphatase Receptor J (PTPRJ) in Normal Mammary Epithelial Cells and Breast Tumors

    PubMed Central

    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.

    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. PMID:22815804

  10. 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.

  11. 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

  12. 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.

  13. 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.

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

    USDA-ARS?s Scientific Manuscript database

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

  15. 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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. 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.

  4. Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

    PubMed

    Boutinaud, Marion; Galio, Laurent; Lollivier, Vanessa; Finot, Laurence; Wiart, Sandra; Esquerré, Diane; Devinoy, Eve

    2013-10-16

    Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

  5. 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

  6. Influence of fatty acid diets on gene expression in rat mammary epithelial cells

    PubMed Central

    Medvedovic, M.; Gear, R.; Freudenberg, J. M.; Schneider, J.; Bornschein, R.; Yan, M.; Mistry, M. J.; Hendrix, H.; Karyala, S.; Halbleib, D.; Heffelfinger, S.; Clegg, D. J.; Anderson, M. W.

    2009-01-01

    Background: This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. Methods: Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. Results: Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Conclusion: Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model. PMID:19351911

  7. 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

  8. 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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  11. 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.

  12. 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.

  13. Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines.

    PubMed

    Rajakylä, Kaisa; Krishnan, Ramaswamy; Tojkander, Sari

    2017-01-01

    Cancer cells are surrounded by a mechanically and biochemically distinct microenvironment that undergoes dynamic changes throughout the neoplastic progression. During this progression, some cancer cells acquire abnormal characteristics that potentiate their escape from the primary tumor site, to establish secondary tumors in distant organs. Recent studies with several human cancer cell lines have shown that the altered physical properties of tumor cells, such as their ability to apply high traction forces to the surroundings, are directly linked with their potential to invade and metastasize. To test the hypothetical interconnection between actomyosin-mediated traction forces and invasion potential within 3D-microenvironment, we utilized two canine mammary tumor cell lines with different contractile properties. These cell lines, canine mammary tumor (CMT)-U27 and CMT-U309, were found to have distinct expression patterns of lineage-specific markers and organization of actin-based structures. In particular, CMT-U309 carcinoma cells were typified by thick contractile actomyosin bundles that exerted high forces to their environment, as measured by traction force microscopy. These high contractile forces also correlated with the prominent invasiveness of the CMT-U309 cell line. Furthermore, we found high contractility and 3D-invasion potential to be dependent on the activity of 5'AMP-activated protein kinase (AMPK), as blocking AMPK signaling was found to reverse both of these features. Taken together, our findings implicate that actomyosin forces correlate with the invasion potential of the studied cell lines.

  14. Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines

    PubMed Central

    Rajakylä, Kaisa; Krishnan, Ramaswamy; Tojkander, Sari

    2017-01-01

    Cancer cells are surrounded by a mechanically and biochemically distinct microenvironment that undergoes dynamic changes throughout the neoplastic progression. During this progression, some cancer cells acquire abnormal characteristics that potentiate their escape from the primary tumor site, to establish secondary tumors in distant organs. Recent studies with several human cancer cell lines have shown that the altered physical properties of tumor cells, such as their ability to apply high traction forces to the surroundings, are directly linked with their potential to invade and metastasize. To test the hypothetical interconnection between actomyosin-mediated traction forces and invasion potential within 3D-microenvironment, we utilized two canine mammary tumor cell lines with different contractile properties. These cell lines, canine mammary tumor (CMT)-U27 and CMT-U309, were found to have distinct expression patterns of lineage-specific markers and organization of actin-based structures. In particular, CMT-U309 carcinoma cells were typified by thick contractile actomyosin bundles that exerted high forces to their environment, as measured by traction force microscopy. These high contractile forces also correlated with the prominent invasiveness of the CMT-U309 cell line. Furthermore, we found high contractility and 3D-invasion potential to be dependent on the activity of 5′AMP-activated protein kinase (AMPK), as blocking AMPK signaling was found to reverse both of these features. Taken together, our findings implicate that actomyosin forces correlate with the invasion potential of the studied cell lines. PMID:28955712

  15. 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

  16. Inhibitory Activity of YKL-40 in Mammary Epithelial Cell Differentiation and Polarization Induced by Lactogenic Hormones: A Role in Mammary Tissue Involution

    PubMed Central

    Scully, Steve; Yan, Wei; Bentley, Brooke; Cao, Qing Jackie; Shao, Rong

    2011-01-01

    We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells. PMID:21991364

  17. 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.

  18. 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

  19. Aluminium-phthalocyanine chloride nanoemulsions for anticancer photodynamic therapy: Development and in vitro activity against monolayers and spheroids of human mammary adenocarcinoma MCF-7 cells.

    PubMed

    Muehlmann, Luis Alexandre; Rodrigues, Mosar Corrêa; Longo, João Paulo Figueiró; Garcia, Mônica Pereira; Py-Daniel, Karen Rapp; Veloso, Aline Bessa; de Souza, Paulo Eduardo Narciso; da Silva, Sebastião William; Azevedo, Ricardo Bentes

    2015-05-13

    Photodynamic therapy (PDT) combines light, molecular oxygen and a photosensitizer to induce oxidative stress in target cells. Certain hydrophobic photosensitizers, such as aluminium-phthalocyanine chloride (AlPc), have significant potential for antitumor PDT applications. However, hydrophobic molecules often require drug-delivery systems, such as nanostructures, to improve their pharmacokinetic properties and to prevent aggregation, which has a quenching effect on the photoemission properties in aqueous media. As a result, this work aims to develop and test the efficacy of an AlPc in the form of a nanoemulsion to enable its use in anticancer PDT. The nanoemulsion was developed using castor oil and Cremophor ELP®, and a monodisperse population of nanodroplets with a hydrodynamic diameter of approximately 25 nm was obtained. While free AlPc failed to show significant activity against human breast adenocarcinoma MCF-7 cells in an in vitro PDT assay, the AlPc in the nanoemulsion showed intense photodynamic activity. Photoactivated AlPc exhibited a 50 % cytotoxicity concentration (CC50) of 6.0 nM when applied to MCF-7 cell monolayers and exerted a powerful cytotoxic effect on MCF-7 cell spheroids. Through the use of spontaneous emulsification, a stable AlPc nanoemulsion was developed that exhibits strong in vitro photodynamic activity on cancer cells.

  20. 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.

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

    USDA-ARS?s Scientific Manuscript database

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

  2. 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.

  3. 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.

  4. 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.

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

    SciTech Connect

    Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.; Opresko, Lee; Coffey, Robert J.; Zangar, Richard C.; Wiley, H. S.

    2008-11-14

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

  6. 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

  7. 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.

  8. The Mammary Epithelial Cell Secretome and its Regulation by Signal Transduction Pathways

    SciTech Connect

    Jacobs, Jon M.; Waters, Katrina M.; Kathmann, Loel E.; Camp, David G.; Wiley, H. S.; Smith, Richard D.; Thrall, Brian D.

    2008-02-01

    Extracellular proteins released by mammary epithelial cells are critical mediators of cell communication, proliferation and organization, yet the actual spectrum of proteins released by any given cell (the secretome) is poorly characterized. To define the set of proteins secreted by human mammary epithelial cells (HMEC), we combined analytical and computational approaches to define a secretome protein set based upon probable biological significance. Analysis of HMEC-conditioned medium by liquid chromatography-mass spectrometry resulted in identification of 889 unique proteins, of which 151 were found to be specifically enriched in the extracellular compartment when compared with a database of proteins expressed in whole HMEC lysates. Additional high mass accuracy analysis revealed 36 proteins whose extracellular abundance increased after treatment with phorbol ester (PMA), a protein kinase C agonist and general secretagogue. Many of the PMA stimulated proteins have been reported to be aberrantly expressed in human cancers and appear to be co-regulated as multigene clusters. By inhibiting PMA-mediated transactivation of the epidermal growth factor receptor (EGFR), a pathway critically required for normal HMEC function, we found that the secretion of specific matrix metalloproteases were also coordinately regulated through EGFR transactivation. This study demonstrates a tiered strategy by which extracellular proteins can be identified and progressively assigned to classes of increasing confidence and regulatory importance.

  9. 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.

  10. Targeting the PyMT Oncogene to Diverse Mammary Cell Populations Enhances Tumor Heterogeneity and Generates Rare Breast Cancer Subtypes

    PubMed Central

    Smith, Brittni A.; Shelton, Dawne N.; Kieffer, Collin; Milash, Brett; Usary, Jerry; Perou, Charles M.; Bernard, Philip S.

    2012-01-01

    Human breast cancer is a heterogeneous disease composed of different histologies and molecular subtypes, many of which are not replicated in animal models. Here, we report a mouse model of breast cancer that generates unique tumor histologies including tubular, adenosquamous, and lipid-rich carcinomas. Utilizing a nononcogenic variant of polyoma middle T oncogene (PyMT) that requires a spontaneous base-pair deletion to transform cells, in conjunction with lentiviral transduction and orthotopic transplantation of primary mammary epithelial cells, this model sporadically induces oncogene expression in both the luminal and myoepithelial cell lineages of the normal mouse mammary epithelium. Microarray and hierarchical analyses using an intrinsic subtype gene set revealed that lentiviral PyMT generates both luminal and basal-like tumors. Cumulatively, these results show that low-level expression of PyMT in a broad range of cell types significantly increases tumor heterogeneity and establishes a mouse model of several rare human breast cancer subtypes. PMID:23486760

  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. Mathematical modeling of mammary ducts in lactating human females.

    PubMed

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

    2015-07-01

    This work studies a 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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?

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

    PubMed

    Boulanger, Corinne A; Bruno, Robert D; Mack, David L; Gonzales, Monica; Castro, Nadia P; Salomon, David S; Smith, Gilbert H

    2013-01-01

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

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

    PubMed

    Maningat, Patricia D; Sen, Partha; Sunehag, Agneta L; Hadsell, Darryl L; Haymond, Morey W

    2007-12-01

    Little is known of the molecular regulation of human milk production because of limitations in obtaining mammary tissue from lactating women. Our objectives were to evaluate whether RNA isolated from breast milk fat globules (MFGs) could be an alternative to mammary biopsies and to determine whether intense breast pumping, which increases prolactin (PRL) secretion, will upregulate alpha-lactalbumin (alpha-LA, a major determinant of lactose synthesis) transcription. RNA was isolated from MFG and transcripts of interest were identified and quantitated by real-time RT-PCR using an external standard for normalization. In addition, we performed microarray studies to determine MFG RNA gene expression profile. Ten lactating women were studied using two protocols: protocol A with intense pumping from 0800 to 0814 h followed by short pumping and protocol B with intense pumping from 1200 to 1214 h preceded by short pumping. Plasma PRL and MFG alpha-LA mRNA expression were measured. During protocol A, plasma PRL (61+/-7-248+/-43 mug/l by 14 min) and alpha-LA (3.5+/-0.9 fold by 6 h; P<0.03) increased. During protocol B, PRL gradually increased over 4 h from 69+/-14 to 205+/-28 mug/l, and further to 329+/-23 mug/l by 12 min of intense pumping; alpha-LA mRNA expression did not increase significantly. We conclude that MFGs provide a unique source to study the in vivo regulation of gene expression in mammary epithelial cells. alpha-LA mRNA is abundant in the MFG and its expression may be regulated by hormonal and temporal factors.

  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. Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK.

    PubMed

    Kim, Ha-Na; Kim, Do-Hee; Kim, Eun-Hee; Lee, Mee-Hyun; Kundu, Joydeb Kumar; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

    2014-08-28

    Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK-IKKβ. Copyright © 2014 Elsevier Ireland Ltd. All rights

  4. 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.

  5. 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.

  6. Wnt Signaling in Mammary Glands: Plastic Cell Fates and Combinatorial Signaling

    PubMed Central

    Alexander, Caroline M.; Goel, Shruti; Fakhraldeen, Saja A.; Kim, Soyoung

    2012-01-01

    The mouse mammary gland is an outstanding developmental model that exemplifies the activities of many of the effector pathways known to organize mammalian morphogenesis; furthermore, there are well-characterized methods for the specific genetic manipulation of various mammary epithelial cell components. Among these signaling pathways, Wnt signaling has been shown to generate plasticity of fate determination, expanding the genetic programs available to cells in the mammary lineage. It is responsible first for the appearance of the mammary fate in embryonic ectoderm and then for maintaining bi-potential basal stem cells in adult mammary ductal trees. Recent technical developments have led to the separate analysis of various mammary epithelial cell subpopulations, spurring the investigation of Wnt-dependent interactions. Although Wnt signaling was shown to be oncogenic for mouse mammary epithelium even before being identified as the principle oncogenic driver for gut epithelium, conclusive data implicating this pathway as a tumor driver for breast cancer lag behind, and we examine potential reasons. PMID:22661590

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  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. 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.

  17. Slug Controls Stem/Progenitor Cell Growth Dynamics during Mammary Gland Morphogenesis

    PubMed Central

    Selmi, Abdelkader; Côme, Christophe; Faraldo, Maria-Luisa M.; Deugnier, Marie-Ange; Savagner, Pierre

    2012-01-01

    Background Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) “master genes”. EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question. Methodology/Principal Findings Using a Slug–lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10–20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres. Conclusions/Significance We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism

  18. 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.

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

    PubMed Central

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

    2016-01-01

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

  1. 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.